Agent for the treatment of alopecia

ABSTRACT

It is an object of the present invention to provide a new agent for the treatment of alopecia, the agent being not only effective and safe for, in particular, alopecia areata, androgenetic alopecia in a male, androgenetic alopecia in a female, female pattern alopecia, postpartum alopecia, seborrheic alopecia, alopecia pityroides, senile alopecia, cancer chemotherapy drug-induced alopecia, and alopecia due to radiation exposure, but also effective for a target having resistance to treatment with minoxidil or finasteride, there being no side effects such as an itching sensation, irritation, or feminization, and no contraindications, the agent suppressing dandruff or having a therapeutic effect for white hair, and the therapeutic effect for alopecia being maintained for a long period even when use of the agent is stopped. 
     The solution means of the present invention is an agent for the treatment of alopecia containing as an active ingredient a C-type natriuretic peptide (CNP), a B-type natriuretic peptide (BNP), a derivative of these NPs, a chimeric peptide of these NPs, or a derivative of a chimeric peptide of these NPs.

RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.13/355,484, filed Jan. 21, 2012, which claims the benefit under 35U.S.C. §119(e) of U.S. provisional application Ser. No. 61/437,032,filed Jan. 28, 2011, the entire disclosures of which are incorporatedherein by reference.

TECHNICAL FIELD

The present invention relates to an agent for the treatment and/orprevention of alopecia, dandruff, white hair, and seborrheic scalp, theagent containing as an active ingredient an A-type natriuretic peptide(ANP), a B-type natriuretic peptide (BNP), a C-type natriuretic peptide(CNP), a derivative of these natriuretic peptides (NPs), a chimericpeptide of 2 or more NPs selected from the above NPs, or a derivative ofa chimeric peptide of the above NPs (hereinafter, they are collectivelycalled ‘natriuretic peptides (NPs)’).

In particular, the present invention relates to an agent for thetreatment and/or prevention of alopecia, dandruff, white hair, andseborrheic scalp, the agent containing as an active ingredient BNP, CNP,a derivative of BNP, a derivative of CNP, a chimeric peptide of CNP orBNP, or a derivative thereof.

The agent for the treatment and/or prevention is expressed simply as anagent for the treatment below.

BACKGROUND ART

1. Alopecia

Alopecia is a disease in which hair is lost. The loss of hair inalopecia is not limited just to head hair but can happen anywhere on thebody. Although not usually life-threatening, since it is accompanied byserious emotional distress due to issues related to appearance, there isa desire for an excellent agent for the treatment and an excellent agentfor the prevention of alopecia. Furthermore, since alopecia is oftenaccompanied by fading of hair color, there is a desire for an agent forthe prevention and an agent for the treatment of fading of hair coloraccompanying alopecia. Moreover, since alopecia is often accompanied bydeterioration of hair quality such as hair becoming finer or hairbecoming shorter, there is a desire for an agent for the prevention andan agent for the treatment of deterioration of hair quality accompanyingalopecia.

With regard to types of alopecia, there are alopecia areata,androgenetic alopecia, postmenopausal alopecia, female pattern alopecia,seborrheic alopecia, alopecia pityroides, senile alopecia, cancerchemotherapy drug-induced alopecia, alopecia due to radiation exposure,trichotillomania, postpartum alopecia, etc.

These types of alopecia have the same symptoms of hair loss, but arebased on different causes, and therapies therefor are different fromeach other. In particular, androgenetic alopecia, which is based on theaction of male hormone, and alopecia areata, which is suspected to be animmune disease, are very different diseases. Furthermore, it is thoughtthat postpartum alopecia, female pattern alopecia, seborrheic alopecia,alopecia pityroides, senile alopecia, cancer chemotherapy drug-inducedalopecia, and alopecia due to radiation exposure have different causesfrom each other, and there are hardly any effective therapies.

Alopecia areata is alopecia in which coin-sized circular to patchy baldarea(s) with a clear outline suddenly occur, without any subjectivesymptoms or prodromal symptoms, etc. in many cases, and subsequentlywhen spontaneous recovery does not occur they gradually increase in areaand become intractable. Alopecia areata is suspected to be an autoimmunedisease, but the cause thereof has not yet been discovered, and there isno known definite treatment method.

Alopecia areata is known to be associated with an autoimmune diseasesuch as a thyroid disease represented by Hashimoto's disease, vitiligo,systemic lupus erythematosus, rheumatoid arthritis, or myasthenia gravisor an atopic disease such as bronchial asthma, atopic dermatitis, orallergic rhinitis.

Androgenetic alopecia (AGA) is alopecia in which male hormone acts onmale hormone-sensitive hair follicles to form vellus hair, and occurs inabout half of males and 10% to 20% of females. It is thought thatgenetic predisposition is a large factor in androgenetic alopecia; inandrogenetic alopecia in males, the head hair on the frontal region andcrown becomes fine and short and turns into vellus hair, the hair lineon the forehead finally retreats, and head hair on the crown is lost. Onthe other hand, in androgenetic alopecia in females, in general thehairline does not change but hair of the entire head, in particular thecrown and the frontal region, becomes fine. Finasteride improves onlyabout ¼ of patients for androgenetic alopecia in males, and sinceadministration of finasteride to females is contraindicated, finasteridecannot be used for androgenetic alopecia in females.

Postpartum alopecia is alopecia in which hair whose growth phase hasbeen maintained by estrogen enters the resting phase all at once due tochildbirth, and hair loss increases. The hair loss of postpartumalopecia usually starts approximately 2 months after childbirth andcontinues until about 6 months after childbirth; since it usuallyrecovers within 1 year unless there is late childbearing, in most casesa treatment is not particularly required, but there are cases in whichhair does not recover spontaneously.

Female pattern alopecia is alopecia that is thought to occur due to adecrease in the amount of the female hormone estrogen relative to theamount of androgen in the bloodstream. It often occurs after themenopause, and in this case it is also called postmenopausal alopecia.Female pattern alopecia might be improved by hormone replacement therapybut is intractable in many cases.

Seborrheic alopecia is alopecia that is caused by excessive sebumsecretion on the scalp, pores are blocked thereby causing inflammationaround the pores or in the hair root, and the hair falls out. Seborrheicalopecia is improved to some extent by removing sebum by washing thehair, but it easily reoccurs and exhibits intractability.

Alopecia pityroides is alopecia that is caused by dandruff blockingpores to thus cause inflammation. Alopecia pityroides is often caused byexcessive hair washing; remission is achieved by reducing the number oftimes of hair washing or using a shampoo having a weak washing power,but it easily reoccurs and is intractable.

Trichotillomania is alopecia due to a hair-pulling disorder.Trichotillomania is a symptom resulting from pathological anxiety andcan be treated by behavioral therapy or psychological therapy.

Senile alopecia is alopecia in which, due to aging regardless of gender,body hair of the entire body, including all of the head hair, graduallybecomes thinner. It is thought that this is a natural phenomenon thatappears in many people due to aging, and this is not particularly atarget for treatment at the present. However, there is a desire forimprovement since there is an increased social requirement for improvingthe quality of life of elderly people accompanying a rise in the averagelifespan.

Cancer chemotherapy drug-induced alopecia is alopecia that is a sideeffect of anticancer treatment with a cancer chemotherapy drug. Theshock to the patient caused by loss of hair in all areas including notonly the head but also the eyebrows, eyelashes, nasal hair, underarmhair, and pubic hair is profound even if it is explained in advance.Since this also hinders the carrying out of cancer chemotherapy, thereis a high need for a treatment for this. Similarly, alopeciaaccompanying radiation exposure is also alopecia that occurs based onthe same mechanism as that for cancer chemotherapy drug-induced alopeciain terms of cancer cells being selectively killed by inhibiting celldivision. Therefore, a drug that can treat alopecia accompanying cancerchemotherapy can also treat alopecia accompanying radiation exposure.

In addition, alopecia due to an adverse reaction to a drug such as anantithyroid drug, an anticoagulant, thallium, a psychotropic drug, or aβ-blocker, alopecia due to a fungus, alopecia due to an endocrinedisorder such as dyspituitarism, hypothyroidism, or hyperthyroidism,alopecia due to a metabolic disorder such as a nutritional disorder,hypoalbuminemia, cachexia, iron-deficiency anemia, zinc deficiency,homocystinuria, or cirrhosis of the liver, toxic alopecia, alopecia dueto high temperature, childbirth, major surgery, sudden body weight loss,or serious illness, etc. are known, and they can be tackled by removingthe respective causes thereof.

Among these types of alopecia, female pattern alopecia, seborrheicalopecia, and alopecia pityroides can be tackled to some extent byremoving the respective causes thereof, but they easily reoccur, and areintractable. Furthermore, although it is thought that the cause offemale pattern alopecia is related to hormone balance, hormonereplacement therapy is indicated for menopausal disorders, osteoporosis,and hyperlipidemia but is not indicated for female pattern alopecia;since there is a possibility of cancer being caused by hormonereplacement therapy, hormone replacement therapy is not carried out forthe purpose of treating female pattern alopecia. Furthermore, withregard to androgenetic alopecia, there is no adequate therapy yet, andwith regard to alopecia areata, even the cause thereof is littleunderstood.

As described above, among the types of alopecia, the types of alopeciathat are difficult to treat are alopecia areata and androgeneticalopecia, and for alopecia areata in particular there are hardly anyeffective treatment methods. Moreover, there are hardly any therapiesfor postpartum alopecia, female pattern alopecia, alopecia pityroides,senile alopecia, and cancer chemotherapy drug-induced alopecia.

2. Androgenetic Alopecia and Methods for its Treatment

The frequency of occurrence of androgenetic alopecia increases with age,and in the case of Japanese people, about 10% of people develop it intheir twenties, 20% in their thirties, 30% in their forties, and about40% in their fifties onward (Non-Patent Document 1). In malehormone-sensitive hair follicles such as in the frontal region, thecrown, etc., in contrast to hair in other areas, the phenomenon ofturning into vellus hair is caused by male hormone to thus cause thehead hair to become thin. Although it is a physiological phenomenon, itssocial effect is large due to the impression of the appearance beinggreatly affected. Recently, a minoxidil external medicine and afinasteride internal medicine, which are effective for androgeneticalopecia, have been developed and have also been actively used in adermatological treatment, but it cannot yet be said that sufficienttherapeutic effects are obtained.

With regard to medicinal agents that can be used in the treatment ofandrogenetic alopecia, there are vasodilators such as minoxidil,carpronium chloride, and various extracts, male hormone activityinhibitors such as finasteride, female hormone drugs such as estrogen,estradiol, and progesterone, and antifungal drugs such as ketoconazole,pentadecane, cytopurine (6-benzylaminopurine), t-flavanone, andadenosine.

In ‘Androgenetic alopecia diagnosis and treatment guidelines, 2010edition’ (Non-Patent Document 2) by the Japanese DermatologicalAssociation, the above-mentioned medicinal agents are evaluated usingfive grades, that is, recommendation A (strongly recommended for use),recommendation B (recommended for use), recommendation C1 (can beconsidered for use, but there is insufficient evidence), recommendationC2 (not recommended because there is no evidence), and recommendation D(recommended not to use). In accordance with the above, as a therapy ofrecommendation A, minoxidil, which is a vasodilator, and finasteride,which is a testosterone 5α-reductase inhibitor, are cited; for minoxidilit is stated that ‘5% minoxidil external solution should be used as thedrug of choice in external therapy for a male case, and 1% minoxidilexternal solution should be used as the drug of choice in treatment fora female case’, and for finasteride internal medicine it is stated that‘it should be used as the drug of choice in internal therapy for a malecase, whereas it should not be used for a female case’. The sameguidelines classify carpronium chloride, pentadecane, cytopurine,t-flavanone, adenosine, and ketoconazole as recommendation C1, which is‘can be considered for use, but there is insufficient evidence’, andclassify cepharanthin as recommendation C2 with a written recommendationsaying ‘better not used’.

Furthermore, minoxidil has side effects such as drug-induced contactalopecia, hair hyperplasia, decrease of blood pressure, and decrease inheart rate, and there is the problem that when its use is stopped thesymptoms recur. With female hormone drugs there is the possibility ofthrombosis. Furthermore, finasteride has side effects such as prostatehyperplasia, erectile dysfunction, and ejaculatory disorder, when itsuse is stopped the symptoms recur, and it is contraindicated forpregnant women.

Furthermore, in accordance with the Drug Interview Form for finasteride(Non-Patent Document 3), the clinical effect of finasteride is confirmedbased on evaluation of photographs of the crown. That is, although ahair thickening effect of finasteride on the crown or O-shaped site ofandrogenetic alopecia has been confirmed, there is no known evidence fora hair thickening effect on hair loss in the frontal region or M-shapedsite. Therefore, there is a desire for a new treatment agent thatexhibits a clear therapeutic effect toward androgenetic alopecia thatcontinues for a certain period after its use is stopped, exhibits cleareffectiveness toward hair loss in areas other than on the crown, and hasfewer side effects.

3. Alopecia Areata and Methods for its Treatment

Alopecia areata is a disease that has the highest frequency amongacquired alopecias; it occurs in about 0.1% to 0.2% of the population inAmerica, and it seems to be at the same level in Japan. Alopecia areatadevelops in a person at any age. A quarter of alopecia areata patientsdevelop it at an age of no older than 15 years old, and it is seenrelatively often among children. Serious types of alopecia areata suchas alopecia totalis or alopecia universalis are relatively often seenamong children. There is no gender difference for alopecia areata. Abouta quarter of alopecia areata patients show characteristic symptoms onthe nails, such as a small dent or a horizontal line. With regard toalopecia areata, basically, the wider the hair loss area, the moreintractable it is, and classification of severity based on the hair lossarea has also been considered (Non-Patent Document 4). Many patientswith alopecia areata do not have a physical disorder other than hairloss, but the patients are deeply worried, and the psychological damageand degradation of QOL are great. Because of this, it is considered tobe a skin disease that must be treated using any possible method, butthere are hardly any effective treatment methods in the currentsituation.

With regard to the clinical classification of alopecia areata, it isclassified according to the number of bald areas, the area, and theconfiguration as follows.

[1]. Standard alopecia areata

Alopecia areata monolocularis: single bald area

Alopecia areata multilocularis: a plurality of bald areas are observed

[2]. Alopecia totalis: bald patch enlarges to the entire head

[3]. Alopecia universalis: hair loss enlarges to the entire body

[4]. Alopecia ophiasis: band-shaped hair loss occurs at the headhairline

Furthermore, as an index representing severity, USA alopecia areataevaluation guidelines determine severity using the proportion (S) ofbald patch area occupying the entire head area and the degree (B) ofhair loss other than on the head. Here, they are defined as follows.

S0: No hair loss

S1: Bald patch is less than 25% of the entire head

S2: Bald patch is 25% to 49%

S3: Bald patch is 50% to 74%

S4: Bald patch is 75% to 99%

S5: 100% hair loss

B0: No hair loss in region other than the head

B1: Partial hair loss is seen in region other than the head

B2: Complete hair loss over whole body

The wider the bald patch area in alopecia areata, the more serious thecase is, and the more intractable it is.

In recent years, the cause of alopecia areata has been thought to be anautoimmune disease of hair follicle tissue (Non-Patent Document 5).There is also a report that the rate of coexistence of alopecia areatawith an atopic disease is relatively high (Non-Patent Document 6). Whenalopecia is caused by exacerbation of atopic dermatitis it is calledatopic alopecia. In particular, atopic alopecia patients having afilaggrin gene abnormality tend to suffer from coexisting seriousalopecia areata (Non-Patent Document 7). Histopathologically also, in agroup having an atopic predisposition, lymphocyte infiltration into anarea around the hair follicle is often seen, and eosinophils and mastcells also infiltrate. Of the infiltrating lymphocytes, CD4-positive Tlymphocytes occupy 60% to 80%, CD8-positive T lymphocytes occupy 20% to40%, and there are a large number of HLA-DR-positive cells andINF-γ-positive cells (Non-Patent Document 8).

However, on the other hand, there is a report that the frequency of anatopic or autoimmune disease in alopecia areata patients is notdifferent from that in healthy people (Non-Patent Document 9).Furthermore, although some relationship between alopecia areata andimmune abnormality has been suggested, the specific causal relationshipis not at all clear.

As medicinal agents that can be used for the treatment of alopeciaareata, there are steroid drugs such as diflorasone, betamethasone,dexamethasone, clobetasol, prednisolone, mometasone, methylprednisolone,Deprodone, difluprednate, fluocinonide, amcinonide, triamcinolone,difluprednate, and hydrocortisone, second-generation antihistamine drugssuch as azelastine, Glycyron (registered trademark), which is a complexof glycyrrhizin, methionine, and glycine, carpronium chloride,cepharanthin, minoxidil, cyclosporin A, Cassia Twig plus Dragon's Boneand Oyster Shell Decoction (Keishikaryukotsuboreito), Pinellia andMagnolia Decoction (Hangekobokuto), biotin, Anthralin (Dithranol),tricyclic antidepressants, etc.

Furthermore, as treatments for alopecia areata there are localimmunotherapy in which a synthetic reagent called SADBE (squalic aciddibutyl ester) or DPCP (diphenylcyclopropenone) is contacted with a hairloss area to thus form a rash in the hair loss area and modulateimmunity, a cooling therapy in which carbon dioxide snow or liquidnitrogen is used, a linearly polarized near-infrared ray irradiationtherapy (Super Lizer therapy), a PUVA therapy, which is a photochemicaltherapy employing in combination Psoralen and long wavelength UV rays(UVA), stellate ganglion block, hypnotherapy, and acupuncture andmoxibustion treatments.

With regard to these medicinal agents and therapies, an evaluation wascarried out in ‘Japanese Dermatological Association Alopecia AreataDiagnosis and Treatment Guidelines 2010 edition’ (Non-Patent Document10) using five categories, that is, recommendation A (stronglyrecommended for use), recommendation B (recommended for use),recommendation C1 (can be considered for use, but there is insufficientevidence), recommendation C2 (not recommended because there is noevidence), and recommendation D (recommended not to use).

In accordance with the above, no therapy is evaluated as recommendationA; as therapies with recommendation B steroid local injection and localimmunotherapy are cited, the steroid local injection ‘should be used formonolocularis and multilocularis adult cases where the state of thedisease is fixed at S1 or below’, and the local immunotherapy ‘should becarried out as the first choice for multilocularis, totalis, anduniversalis cases regardless of age where the state of the disease isfixed at S2 or above’. As therapies with recommendation C1, a steroidpulse therapy by intravenous drip infusion is for ‘adult cases of S2 orabove rapidly progressing within 6 months after onset’, orallyadministered steroid or orally administered steroid pulse therapy is for‘adult cases of S2 or above in which hair loss is rapidly progressing’,and the second-generation antihistamine drug is ‘one of the combinedtherapies for monolocularis and multilocularis cases having an atopicpredisposition’, all being recommended for use with recommendation C1.The same guidelines classify cepharanthin, Glycyron, steroid externalpreparation, carpronium chloride external preparation, minoxidilexternal preparation, cooling therapy, linearly polarized near-infraredray irradiation therapy, and PUVA therapy as recommendation C1 ‘whiletaking the actual results of diagnosis and treatment into consideration’even though the ‘benefit is not sufficiently proved at the presentstage’.

On the other hand, cyclosporin A and Keishikaryukotsuboreito areclassified as recommendation C2 with a written recommendation of ‘cannotbe recommended at the current time’. Furthermore, Anthralin, a tricyclicantidepressant, stellate ganglion block, and hypnotherapy are classifiedas recommendation C2 with a written recommendation of ‘better not used’or ‘better to withhold’. The acupuncture and moxibustion treatments areclassified as recommendation D since they have ‘not reached a standardfor medical evaluation’.

With regard to the prognosis for alopecia areata, in a case where theduration of the bald patch is long or a case where there is a history ofan atopic disease or an autoimmune endocrine disease, the possibility ofa cure is low (Non-Patent Document 11). In accordance with reports froma number of European and American institutions, of all patients, 34% to50% shift to the totalis type or the universalis type, and in thesecases the recovery rate becomes as low as no greater than 10%(Non-Patent Document 12). In adult cases where the hair loss area isless than 50%, 56% thereof recovered, but for alopecia areata with ahair loss area of 50% or greater the recovery rate was only 3.7%(Non-Patent Document 13). Furthermore, the recovery rate is also low forcases in which it developed at the age of 15 or younger or for alopeciaophiasis (Non-Patent Document 12), and there is no recommendabletherapy. Therefore, there is a strong desire for development of a newtherapy for serious cases of alopecia, in particular S2 or above.

4. Female Pattern Alopecia and Methods for its Treatment

In female pattern alopecia, loss of hormone balance causes a shorterhair growth phase and a longer resting phase. Because of this, thenumber of hairs emerging from one pore decreases, the hair itselfbecomes half or less the width of the healthy state, or the hair colorbecomes pale. As a result, a state in which the scalp is seen throughhairs around the center of the crown, that is, a pattern in which itbecomes thinner overall, is observed.

Olsen E. et al. 2003 (Non-Patent Document 25) state that the manner ofwidening of the hair parting from the crown toward the frontal regionresembling the manner of spreading of the branches of a Christmas treeis an important initial symptom for female pattern alopecia. Femalepattern alopecia usually has an older age of onset than that ofandrogenetic alopecia, and is often confirmed from the 40s to 50s orafter menopause. Since oral administration of finasteride, whichsuppresses activation of testosterone into dihydrotestosterone, is noteffective for menopausal females, it is now generally thought thatfemale thinning hair is not the same as androgenetic alopecia. As hereindescribed, female pattern alopecia is diagnosed, clearly separately fromandrogenetic alopecia, from gender, age, and a diffuse alopecia state.

With regard to the causes of female pattern alopecia, it is caused by adecrease in estrogen due to the menopause as well as a decrease infemale hormone due to severe diet, stress, or suspension of oralcontraceptive use.

With regard to treatment for female pattern alopecia, finasteride iscontraindicated in practice since it cannot be used for female patternalopecia of a female who might be pregnant or a female who isbreast-feeding. Minoxidil and cepharanthin both have a weak effect onfemale pattern alopecia. There is no known effect of female hormonereplacement therapy on female pattern alopecia. As described above,there are hardly any effective treatment methods for female patternalopecia.

5. Postpartum Alopecia and Methods for its Treatment

Many females experience the hair becoming thick during pregnancy andhair loss occurring when breast-feeding after childbirth. Since thecause of postpartum alopecia is clear, its diagnosis is easy and itcures spontaneously in many cases, it is not particularly treated.However, it is said that about 40% of postnatal females experiencepostpartum alopecia, so there are a considerable number of patients.Furthermore, there are variations among individuals for the degree ofhair loss of postpartum alopecia and the extent of recovery, and thereare rare cases in which hair loss does not stop if postpartum stresscontinues. In these cases, if a steroid is used for a long period, itoften becomes difficult for hair to grow, and this requires attention.

As described above, when postpartum alopecia does not spontaneouslyrecover, a steroid cannot be used for the treatment thereof, and thereare no other effective therapies.

6. Seborrheic Alopecia and Methods for its Treatment

In the case of seborrheic alopecia, a large amount of sebum iscontinuously secreted from the pores, and the pores are blocked to sucha degree that it can be seen by the naked eye. Because of this, if sebumis removed, the pores are seen to be red due to inflammation, thisinflammation causes hair to be lost, and diagnosis is thereforerelatively easy.

With regard to the cause of seborrheic alopecia, it is thought thatinflammation is caused by abnormal growth of indigenous bacteria on thescalp due to excessive secretion of sebum; a certain therapeutic effectcan be anticipated by appropriately removing sebum in the pores using aless irritating shampoo, but it is difficult to maintain an appropriatelevel of sebum. It is also said that proliferation of Malasseziaglobosa, which is a type of fungus, is one factor; there are cases inwhich external application of an antifungal agent shows a therapeuticeffect, but since constitutional secretion of excess sebum is theultimate factor, it is difficult to improve, and there are hardly anytreatments for seborrheic alopecia.

7. Alopecia Pityroides and Methods for its Treatment

Alopecia pityroides is a disease in which a large amount of dandruff isgenerated abnormally such that dandruff becomes scab-like and blocks thepores to thus cause inflammation, and the diagnosis thereof isrelatively easy. With regard to the cause thereof, it is said thatindigenous bacteria on the scalp proliferate abnormally due to anabnormal hormone balance, and this gives rise to hair loss.

As a treatment therefor, a steroid treatment exhibits the highest effectamong present therapies, but since this method might take a long time toachieve a complete cure or might easily result in a case in which thesymptoms become chronic and the treatment becomes rather difficult, itis not a therapy that can be recommended. However, since there is noother therapy in the current situation, in general, the shampoo ischanged to one having a weaker washing power, the number of times ofhair washing is decreased, or a moisturizer is applied to thus preventthe scalp from becoming dry, with the expectation that the symptoms willbe alleviated.

8. Senile Alopecia and Methods for its Treatment

Senile alopecia is alopecia in which, regardless of difference ingender, hair falling out and thinning occur for body hair of the entirebody, including the entire head, accompanying aging. As symptoms ofsenile alopecia, the characteristics of the scalp becoming dry and bloodvessels being visible through the skin are often observed. The cause ofsenile alopecia is due to deterioration in the ability to produce newcells in the body caused by aging, and it is therefore difficult totreat. As a current treatment for senile alopecia, hair papilla areactivated by massaging the scalp, etc. to thus improve the possibilityof hair growth, and there are hardly any therapies.

9. Cancer Chemotherapy Drug-Induced Alopecia and Methods for itsTreatment

Cancer chemotherapy drug-induced alopecia is alopecia that occurs due toa cancer chemotherapy drug inhibiting cell division to thus selectivelykill cancer cells, which actively undergo cell division, and at the sametime inhibiting hair matrix cells, which also actively undergo celldivision in the same manner.

It is known that alopecia is caused by chemotherapy agents such as, forexample, cyclophosphamide, ifosfamide, doxorubicin, amrubicin,paclitaxel, docetaxel, irinotecan, epirubicin, etoposide, actinomycin D,bleomycin, vincristine, vinorelbine, carboplatin, methotrexate,cisplatin, melphalan, fluorouracil, gemcitabine, capecitabine,tegafur-gimeracil-oteracil potassium, vinblastine, and ixabepilone,which are generic names.

In general, with regard to cancer chemotherapy drug-induced alopecia,taking the initial administration of a chemotherapy drug as the startingpoint, hair loss starts after 10 days, hair loss becomes conspicuousafter 20 days, and all body hair is lost after 30 to 60 days. Withregard to recovery of body hair after completion of cancer chemotherapy,taking completion of administration of the chemotherapy drug as thestarting point, hair growth normally starts 3 to 6 months later, andbody hair has almost recovered in about 8 to 12 months.

However, since cancer chemotherapy drug-induced alopecia is caused byhair matrix cells within the hair follicle being inhibited by the cancerchemotherapy drug, recovery of body hair after completion of the cancerchemotherapy varies depending on the degree of inhibition of hair matrixcells. Because of this, although there seems to be recovery, even in amild case the quality of the hair might be degraded and the color orthickness of the hair might change, and in serious cases recovery mightbe only to the growth of downy hair. Furthermore, even if body hairrecovers, since a state in which there is no body hair continues forhalf a year to one and a half years, the appearance changes greatlyduring this period and this, coupled with anxiety with respect to thecancer itself, causes considerable distress to the patient.

Objective evaluation of cancer chemotherapy drug-induced alopecia isgenerally carried out as part of a skin disorder and hair lossevaluation based on Common Terminology Criteria for Adverse Events(CTCAE). Specifically, evaluation by CTCAE of cancer chemotherapydrug-induced alopecia is carried out such that a case without hair lossis evaluated as grade 0, a case with light hair loss is grade 1, and acase with conspicuous hair loss is grade 2.

There is no method for preventing or treating cancer chemotherapydrug-induced alopecia. Therefore, until cancer chemotherapy drug-inducedalopecia spontaneously recovers, countermeasures such as a wig beingused in place of head hair, eyebrows being drawn by an eyebrow pencil,and artificial eyelashes being used are taken to give a naturalappearance.

As described above, alopecia areata, androgenetic alopecia, femalepattern alopecia, postpartum alopecia, seborrheic alopecia, alopeciapityroides, senile alopecia, and cancer chemotherapy drug-inducedalopecia not only have different causes from each other, but thetherapies therefor are also very limited. For example, there are hardlyany therapies for female pattern alopecia, postpartum alopecia, alopeciapityroides, senile alopecia, and cancer chemotherapy drug-inducedalopecia. Moreover, with regard to androgenetic alopecia, a treatmentusing minoxidil or finasteride is recommended, but the effects thereofare weak.

Furthermore, for alopecia areata, steroid local injection or localimmunotherapy are recommended, and although there are cases in whichsome effects can be seen, there are many cases in which no effects canbe seen at all, and there are cases in which hair grows temporarily butwhen treatment is stopped the situation deteriorates due to steroidrebound. There is therefore a strong desire for the development of newtherapies therefor.

4. Natriuretic Peptides

As natriuretic peptides (NPs), a family of 3 types of natriureticpeptides is known; specifically there are atrial natriuretic peptide(ANP; atrial natriuretic peptide), B-type natriuretic peptide (BNP;B-type natriuretic peptide), and C-type natriuretic peptide (CNP; C-typenatriuretic peptide), those formed from 28, 32, and 22 amino acidresidues respectively being the ones that are best known.

(1) ANP and BNP

ANP is mainly synthesized in the atrium and BNP is mainly synthesized inthe ventricle, and they are then secreted from the heart to the wholebody. Substantially 100% of ANP and BNP circulating in the blood is saidto be heart-derived. It is reported that ANP and BNP are deeply involvedin clinical conditions such as high blood pressure, cardiomegaly, heartfailure, myocardial infarction, valvular disease, arrhythmia, andpulmonary hypertension.

Human ANP is a peptide formed from 28 amino acids produced in andsecreted from the atrial cells, and forms a cyclic structure due to anintramolecular disulfide bond between cysteine residue 7 and cysteineresidue 23. ANP exhibits a diuretic action in the kidney, and it relaxesand expands vascular smooth muscle in blood vessels. On the other hand,human BNP is a peptide formed from 32 amino acids produced in andsecreted from the ventricular cells, and forms a cyclic structure due toan intramolecular disulfide bond between cysteine residue 10 andcysteine residue 26. BNP also has a diuretic action and a vasodilatingaction. BNP is a peptide that was isolated from pig brain in 1988 andidentified, and is also called brain natriuretic peptide.

Both ANP and BNP bind to an NPR-A receptor having a guanylate cyclasedomain (also called GC-A), promote the production of cGMP, and exhibitthe above-mentioned action. In reality, secretion of ANP is promotedaccompanying an increase in atrial inflation pressure in congestiveheart failure, etc., and it functions to alleviate the symptoms ofcongestive heart failure, etc. by virtue of the above-mentioned action.Secretion of BNP is also promoted in myocardial infarction, etc., and itfunctions to alleviate various symptoms accompanying myocardialinfarction, etc. by virtue of the above-mentioned action (Non-PatentDocument 14). Most BNP in the blood is derived from the ventricle, butsome is also secreted from the atrium. Expression of both ANP and BNP isincreased to 100 times the normal level in a heart failure state, but itis also reported that the increase of BNP is larger and faster than thatof ANP. ANP (hANP) is commercially available in Japan as an acute heartfailure treatment drug, and in the USA BNP is commercially available asa congestive heart failure treatment drug.

(2) CNP

Because CNP was first discovered in the brain, it was thought that itfunctioned as a cranial nerve peptide, but it was later found that italso exists in the periphery. In particular, since in the blood vesselwall there are many CNP-specific receptors in smooth muscle cells,monocyte/macrophage cells and endothelial cells produce CNP, etc., it isthought that CNP is involved in the suppression of growth of smoothmuscle cells as a local factor in the blood vessel wall. Because ofthis, the possibility of administration of CNP being able to preventintravascular restenosis, which occurs with a certain frequency amongpatients with an ischemic heart disease after receiving percutaneoustransluminal coronary angioplasty (PTCA) and is a clinical problem, iscurrently being examined in terms of clinical application.

Furthermore, an animal experiment has recently been reported in whichintravenous administration of CNP clearly improves enlargement of theheart and fibrosis after myocardial infarction and improves the cardiacfunction. Fibrosis of the heart is known to cause diastolic heartfailure or arrhythmia, and since CNP has the action of stronglysuppressing the growth of fibroblasts, research has been carried outinto its use as a drug for the treatment of fibrosis of the heart. SinceCNP is a hormone present in the body, there are no concerns about sideeffects, and its application as a clinical treatment drug forarteriosclerotic disease or cardiac disease is anticipated. As CNP,CNP-22 having 22 amino acids, CNP-53 having 53 amino acids in whichamino acid residues are added to the N terminal of the above, etc. areknown.

(3) Natriuretic Peptide Receptors

As receptors for NPs, three types are known, that is, an NPR-A receptorhaving a guanylate cyclase domain (also called GC-A), an NPR-B receptorhaving a guanylate cyclase domain (also called GC-B), and an NPR-Creceptor having no guanylate cyclase domain, and it is known that ANPbinds to the NPR-A receptor and the NPR-C receptor, BNP binds to theNPR-A receptor and the NPR-C receptor, and CNP binds to the NPR-Breceptor and the NPR-C receptor.

It is said that activation of the NPR-A receptor brings aboutvasodilatory action, diuretic action, and cytostatic action. On theother hand, NPR-B receptors are present in large numbers in vascularsmooth muscle cells, and are thought to bring about growth-inhibitionaction for vascular smooth muscle cells. The NPR-C receptor is alsocalled a clearance receptor and is though to remove NPs in blood andregulate the concentration of NPs in tissue.

(4) Relationship Between Natriuretic Peptides and the Immune System

With regard to natriuretic peptides, historically ANP was firstdiscovered as a peptide secreted by the atrium, and its vasodilatoryaction and diuretic action have received attention. Following this, BNPand CNP were found as peptides analogous to ANP. Due to such historicalreasons, with regard to the relationship between natriuretic peptidesand immunity, those related to the cardiovascular system have attractedattention.

Furthermore, following this, because of poor growth of the cartilage ithas been found that a CNP knockout mouse exhibits a microsomia-likephenotype (Non-Patent Document 15), and since CNP is anticipated topromote regeneration of articular cartilage, the relationship betweenarthritis and natriuretic peptides is also attracting attention.However, there is no mention in this publication of a relationshipbetween CNP and alopecia.

It has been suggested that ANP has a role in arthritis or septicemiasince it suppresses secretion by macrophages of tumor necrosis factor α(INF-α) and interleukin 1β (IL1β), which are inflammatory cytokines(Non-Patent Document 16). However, there is no mention in thispublication of a relationship between ANP and alopecia.

Furthermore, since it has been reported that the concentration of BNP inblood increases in parallel to heart transplant rejection, it has beensuggested that it is involved in immune modulation in the cardiovascularsystem (Non-Patent Document 17). However, there is no mention in thispublication of a relationship between BNP and alopecia.

Kuroski de Bold et al. have examined the immunomodulating action ofnatriuretic peptides while noting an increase in the concentration ofBNP in blood at the time of heart transplant rejection and have foundthat ANP and BNP both suppress lymphocyte proliferation (Non-PatentDocument 18). However, there is no mention in this publication of arelationship between NP and alopecia.

On the other hand, Chiurchiu et al. have examined the immunomodulatingaction of BNP while noting the relationship with cardiac disease andsepticemia and have reported that since BNP promotes the release bymacrophages of arachidonic acid, prostaglandin E2 (PGE2), andleucotriene B4 (LTB4), which are inflammatory cytokines, and interleukin10 (IL10), which is an anti-inflammatory cytokine, it has some type ofaction in modulating inflammation, but they could not conclude whetheror not it functions to suppress or promote inflammation overall(Non-Patent Document 19). There is no mention in this publication eitherof a relationship between BNP and alopecia.

It has been reported that CNP is secreted from macrophages (Non-PatentDocument 20). Furthermore, Scotland et al. have reported that CNPsuppressed platelet aggregation and leucocyte migration during thecourse of examining the role of CNP in myocardial damage after cardiacischemia and reperfusion (Non-Patent Document 21). However, there is nomention in these publications of a relationship between CNP andalopecia.

Similarly, Obata et al. have examined the action of CNP in myocarditisand have reported that when pig myosin was injected into a ratmyocarditis model and CNP was administered continuously for 1 weekfollowing this, necrosis and inflammation of the heart tissue weresuppressed, angiogenesis was promoted, and deterioration of cardiacfunction was suppressed (Non-Patent Document 22). However, there is nomention in this publication of a relationship between CNP and alopecia.

Furthermore, since CNP knockout mice show a microsomia-like phenotype,CNP is attracting attention in terms of the relationship with the growthof cartilage. Agoston et al. have found that, in primary culture ofcartilage cells separated from mouse embryo neckbone, dexamethasoneincreases expression of the CNP gene (Non-Patent Document 23). However,there is no mention in this publication of a relationship between CNPand alopecia.

As hereinbefore described, in recent years, the relationship betweenimmunity and natriuretic peptides has been attracting attention, but itis only the relationship between cardiovascular inflammation andnatriuretic peptides or between arthritis and natriuretic peptides thatis attracting attention, and there is no report of a relationshipbetween alopecia and natriuretic peptides.

(5) Reports on Applications Related to Natriuretic Peptides

There are a large number of reports on the application of ANP, BNP, andCNP as described below in addition to the above. However, none of thesepublications refer to a relationship between natriuretic peptides andspecific types of alopecia such as alopecia areata, androgeneticalopecia, alopecia pityroides, postpartum alopecia, female patternalopecia, seborrheic alopecia, trichotillomania, and senile alopecia.Furthermore, none of these publications demonstrate that CNP or BNP isuseful for the treatment of alopecia.

Shoji Tanaka et al. have proposed C-type natriuretic peptides exhibitinggrowth-inhibitory action for vascular smooth muscle cells and a vascularsmooth muscle growth inhibitor containing these peptides as an activeingredient (Patent Document 1).

However, this means the use of CNP as an agent for the inhibition ofsmooth muscle cells and does not suggest the application of CNP or BNPto an agent for the treatment of alopecia.

Katsuhiko Nakata et al. have proposed an eye dropper for the promotionof lacrimal secretion or the treatment of keratoconjunctival disorderthat contains a natriuretic peptide as an active ingredient, and ANP,BNP, and CNP are cited as natriuretic peptides that can be used (PatentDocument 2).

However, this concerns utilization of the action of ANP, BNP, and CNP inpromotion of lacrimal secretion as an eye dropper for the treatment ofkeratoconjunctival disorder, and does not suggest the application of CNPor BNP to an agent for the treatment of alopecia.

Kazuwa Nakao et al. have proposed a composition for increasing heightthat is administered to an individual having no FGFR3 abnormality andcontains a guanyl cyclase B (GC-B) activator such as CNP as an activeingredient (Patent Document 3).

However, the intention was to utilize CNP as a composition forincreasing height based on the knowledge that the length between thenose and the anus is larger for a transgenic mouse overexpressing CNPthan for a normal littermate, and does not suggest any application ofCNP or BNP to an agent for the treatment of alopecia.

Similarly, Kazuwa Nakao et al. have proposed an agent for the treatmentor prevention of arthritis, the agent containing a guanyl cyclase B(GC-B) activator such as CNP as an active ingredient (Patent Document4).

However, this is only a finding that in a transgenic mouseoverexpressing CNP, the thickness of the articular cartilage is largecompared with a normal littermate, and when CNP is continuouslyadministered to an arthritis model animal, the arthritis is suppressed,the intention being to utilize CNP as an agent for the treatment orprevention of arthritis, and does not suggest the application of CNP orBNP to an agent for the treatment of alopecia.

Similarly, Kazuwa Nakao et al. have proposed an agent for the preventionor treatment of fatty liver that contains a GC-A receptor agonist as anactive ingredient since in a BNP transgenic mouse overexpressing BNPthere is resistance to fat increasing with a high-fat diet load and theglucose tolerance and insulin sensitivity are improved, and a heteroknockout mouse with respect to a gene for GC-A receptor, which is areceptor for ANP and BNP, easily becomes fat with a high-fat diet load,and the glucose tolerance and insulin sensitivity are impaired (PatentDocument 6).

However, this does not suggest the application of a natriuretic peptideto an agent for the treatment of alopecia and does not mention CNP atall.

Isao Sakaida et al. have proposed an agent for suppressing hepaticfibrosis that contains a natriuretic peptide as an active ingredientbased on the finding that when ANP or CNP was continuously administeredto rats together with N-diethylnitrosoamine, hepatic fibrosis due toN-diethylnitrosoamine was suppressed (Patent Document 7).

However, the intention was to use a natriuretic peptide for preventionof cirrhosis of the liver or liver cancer, and there was no suggestionof the application of a natriuretic peptide to an agent for thetreatment of alopecia.

Yasuhiko Ito et al. have proposed an agent for suppressing peritonealfibrosis that contains a natriuretic peptide as an active ingredientbased on the finding that when ANP was continuously administered to arat abrasion-induced peritoneal fibrosis model, the peritoneal fibrosiswas suppressed (Patent Document 8).

However, the intention was to use a natriuretic peptide for suppressionof peritoneal fibrosis at the time of peritoneal dialysis and there wasno suggestion of the application of a natriuretic peptide to an agentfor the treatment of alopecia.

Chen et al. have proposed a peritoneal dialysis solution containing ANPbased on the finding that when a peritoneal dialysis solution containingANP was injected into a rat, net ultrafiltration and sodium clearanceincreased (Patent Document 9).

However, the intention was to add ANP to a peritoneal dialysis solutionand apply it to the treatment of a patient with kidney damage, and therewas no suggestion of the application of a natriuretic peptide to anagent for the treatment of alopecia.

Toshiyuki Hori et al. have proposed an agent for the prevention ortreatment of a Th1 immune disease that contains a GC-A receptor agonistas an active ingredient based on the finding that when LPS is added to amedium the proliferation of naive T cells induced by dendritic cells wassuppressed by the simultaneous addition of ANP (Patent Document 10).

However, the intention was to apply ANP to the prevention or treatmentof a Th1 immune disease such as Crohn's disease, and there was nosuggestion of the application of a natriuretic peptide to an agent forthe treatment of alopecia.

Hisako Koide et al. have proposed a preparation for repair andregeneration of tissue and organ that contains as an active componentANP, BNP, CNP, Urodilatin (P-Uro), a precursor thereof, a derivativethereof, or a combination thereof, and which may contain apharmaceutically acceptable diluent, excipient, filler, or adjuvant, andhave cited, as one example of the repair and regeneration of tissue andorgan, hair restoration, hair growth, and hair thickening (PatentDocument 5). Furthermore, in Example 3 of the same publication, it wasreported that when ANP was applied twice a day after hair washing to thescalp of ‘54 year old and 89 year old males with thin head hair’, ‘downyhair-like hair growth was observed on the frontal region with 1 week ofANP administration, and sites started to appear where black hair papillaappeared after hair had fallen out’, ‘after 2-3 weeks, resilience andrigidity increased for the entire head hair’, and ‘after 1 month thescalp, which could been seen before use, was obviously difficult tosee’.

However, Patent Document 5 describes only hair growth due to ANP anddoes not demonstrate any of hair restoration, hair growth, and hairthickening by CNP or BNP. Furthermore, with regard to alopecia, thereare various types of alopecia in terms of the cause, and their therapiesare very different from each other, but the mere description of ‘54 yearold and year old males with thin head hair’ cannot clarify for whichtype of alopecia an effect was exhibited; since the characteristic that‘the foremost line of retreating head hair moved forward’ is acharacteristic that can be seen in various types of alopecia, it is notclear what kind of cause or disease the ‘thin hair’ is derived from.Furthermore, said publication only considers a ‘drug for hair growth,hair restoration, and hair thickening’ as a very broad concept; it isunclear whether it is a treatment agent or a preventive agent, and itdoes not suggest a treatment or preventive agent for a specific alopeciaand does not suggest a treatment or preventive agent for dandruff, whitehair, and seborrheic scalp. It is therefore clear that this publicationdoes not suggest an agent for the treatment or prevention of alopeciaareata, androgenetic alopecia, postpartum alopecia, female patternalopecia, seborrheic alopecia, alopecia pityroides, senile alopecia,cancer chemotherapy drug-induced alopecia, alopecia due to radiationexposure, dandruff, white hair, and seborrheic scalp.

Shoji Tanaka et al. have announced as described below that CNP has acompletely different structure and effect from those of ANP and BNP(Patent Document 1).

‘It is currently thought that ANP and BNP both function as a hormonesecreted from the heart into the blood, also function as aneurotransmitter, and play an important role in maintaining homeostasisin the fluid volume and blood pressure of a living body . . . there aremany unclear points regarding the physiological role of CNP as an NP.That is, the amino acid primary sequence of CNP is similar to those ofANP and BNP; furthermore, it exhibits natriuretic action and hypotensiveaction by in vivo administration, and it is therefore classed asbelonging to the NP family. However, since the natriuretic action andhypotensive action of CNP are much weaker than those of ANP and BNP (1/50 to 1/100) . . . . CNP occupies a specific position within the NPfamily, and it is assumed that with regard to its physiological role, itmight play a role other than maintenance of homeostasis in the fluidvolume and blood pressure . . . when the structure of CNP is comparedwith those of ANP and BNP, it can be seen that CNP is different from ANPor BNP in terms of the points described below . . . . That is, it can beseen that the amino acid primary sequence of CNP is completely differentfrom that of ANP or BNP for the exocyclic N-terminal domain, and within17 amino acid residues in the endocyclic domain it is different from ANPby 5 residues and from BNP by 4 residues. Furthermore, the structure ofthe exocyclic C-terminal domain of CNP is very different from those ofANP and BNP, CNP does not have a tail structure, which is present in ANPand BNP (in the case of ANP and BNP, there are 5 amino acid residuesadded to the C-terminal of the cyclic structure for ANP and 6 amino acidresidues for BNP, and these structures are called tail structures forconvenience). It is clear that the above-mentioned difference instructure between CNP and ANP or BNP is involved in exhibition of theabove-mentioned characteristic pharmacological action of CNP.’

In reality, as shown in FIG. 1 also, ANP, BNP, and CNP have verydifferent structures and are thought to carry out different roles fromeach other.

RELATED ART DOCUMENTS Patent Documents

-   [Patent Document 1] JP, A, 6-9688-   [Patent Document 2] JP, A, 2000-169387-   [Patent Document 3] WO2005/094890-   [Patent Document 4] WO2005/094889-   [Patent Document 5] JP, A, 2008-162987-   [Patent Document 6] Republished Japanese translation 2008-032450 of    a PCT application-   [Patent Document 7] JP, A, 2010-168283-   [Patent Document 8] Republished Japanese translation 2008-140125 of    a PCT application-   [Patent Document 9] Published Japanese translation 2000-516836 of a    PCT application-   [Patent Document 10] Republished Japanese translation 2004-110489 of    a PCT application

Non-Patent Documents

-   [Non-Patent Document 1] Satoshi Itami: Hair survey (male pattern    hair loss) Japanese adult males, The Japan Medical Journal: No.    4209, 27-29, 2004-   [Non-Patent Document 2] Androgenetic alopecia diagnosis and    treatment guidelines (2010 edition) (The Japanese Journal of    Dermatology: 120(5), 977-986, 2010)-   [Non-Patent Document 3] Drug Interview Form Propecia tablets,    revised July 2011, MSD-   [Non-Patent Document 4] Olsen E et al.: Alopecia areata    investigational assessment guidelines, J Am Acad Dermatol 40:    242-246, 1999-   [Non-Patent Document 5] Gilhar A et al.: Autoimmune hair loss    (alopecia areata) transferred by T lymphocytes to human scalp    explants on SCID mice, J Clin Invest. 101: 62-67, 1998-   [Non-Patent Document 6] Shellow W V et al.: Profile of alopecia    areata: a questionnaire analysis of patient and family, Int J    dermatol 31: 186-189, 1992.-   [Non-Patent Document 7] Betz R C et al., Loss-of-function mutations    in the filaggrin gene and alopecia areata: strong risk factor for a    severe course of disease in patients comorbid for atopic disease, J    Invest Dermatol. 127, 2539-2543, 2007.-   [Non-Patent Document 8] Kensei Katsuoka: Atopic disease and alopecia    areata—atopic alopecia areata—, Derma 23, 9-12, 1999-   [Non-Patent Document 9] Serarslan G et al., Is atopy and    autoimmunity more prevalent in patients with alopecia areata? A    comparative study, J Eur Acad Dermatol Venereol. doi:    10.1111/j.1468-3083.2011.-   [Non-Patent Document 10] Japanese Dermatological Association    alopecia areata diagnosis and treatment guidelines 2010 (The    Japanese Journal of Dermatology: 120(9), 1841-1859, 2010)-   [Non-Patent Document 11] Ikeda T.: A new classification of alopecia    areata, Dermatologica 131: 421-445, 1965.-   [Non-Patent Document 12] MacDonald Hull S P et al.: Guidelines for    the management of alopecia areata, Br J Dermatol 149: 692-699, 2003-   [Non-Patent Document 13] Tosti A et al.: Alopecia areata: A long    term follow-up study of 191 patients, J Am Acad Dermatol 55:    438-441, 2006-   [Non-Patent Document 14] Yoshibayashi M. et al., Brain natriuretic    peptide versus atrial natriuretic peptide—physiological and    pathophysiological significance in children and adults: a review,    Eur J Endocrinol. 135(3): 265-8, 1996-   [Non-Patent Document 15] Chusho H. et al., Dwarfism and early death    in mice lacking C-type natriuretic peptide, Proceedings of the    National Academy of Sciences of the United States of America, 98(7):    4016-21, 2001-   [Non-Patent Document 16] Kiemer A. K., Vollmar A. M., Annals of the    Rheumatic Disease, The atrial natriuretic peptide regulates the    production of inflammatory mediators in macrophages, 60:    iii68-iii70.2001-   [Non-Patent Document 17] Meirovich Y. F. et al., Relationship    between natriuretic peptides and inflammation: proteomic evidence    obtained during acute cellular cardiac allograft rejection in    humans, the Journal of Heart and Lung Transplantation, 27(1): 31-7,    2008-   [Non-Patent Document 18] de Bold M L et al., Cardiac hormones ANF    and BNP modulate proliferation in the unidirectional mixed    lymphocyte reaction, the Journal of Heart and Lung Transplantation,    29(3): 323-6, 2010-   [Non-Patent Document 19] Chiurchiù V. et al., Brain Natriuretic    Peptide (BNP) regulates the production of inflammatory mediators in    human THP-1 macrophages, Regulatory Peptides, 148(1-3): 26-32, 2008-   [Non-Patent Document 20] Kubo A et al, C-type natriuretic peptide is    synthesized and secreted from leukemia cell lines, peripheral blood    cells, and peritoneal macrophages, Experimental Hematology, 29(5):    609-15, 2001-   [Non-Patent Document 21] Scotland R. S. et al., C-type natriuretic    peptide inhibits leukocyte recruitment and platelet-leukocyte    interactions via suppression of P-selectin expression, Proceedings    of the National Academy of Sciences, 102(40): 14452-7, 2005-   [Non-Patent Document 22] Obata H. et al., CNP infusion attenuates    cardiac dysfunction and inflammation in myocarditis, Biochemical and    Biophysical Research Communications, 356(1): 60-6, 2007-   [Non-Patent Document 23] Agoston H. et al., Dexamethasone stimulates    expression of C-type Natriuretic Peptide in chondrocytes, BMC    Musculoskeletal Disorders, 7: 87, 2006-   [Non-Patent Document 24] Norwood O T. Male pattern baldness:    Classification and incidence, South Med. J. 68: 1359-70, 1975-   [Non-Patent Document 25] Olsen E A.: Current and novel methods for    assessing efficacy of hair growth promotors in pattern hair loss, J    Am Acad Dermatol; 48: 253-62, 2003

SUMMARY OF THE INVENTION Problems to be Solved by the Invention

In spite of the fact that alopecia causes serious emotional distress dueto problems related to appearance, no definite therapy has yet beenfound. With regard to androgenetic alopecia in particular, evenminoxidil or finasteride cannot give a sufficient therapeutic effect inmany cases, and with regard to alopecia areata, there are hardly anyeffective treatment methods. Furthermore, since alopecia areata is arecurring disease and also an intractable disease, greatly degrades theappearance, and might cause depression, it is therefore necessary for itto be treated.

With regard to postpartum alopecia, after childbirth, since a largeamount of body hair is lost in a short period, coupled with postpartumdepression the emotional burden on the patient is large, but there arehardly any therapies. Female pattern alopecia, seborrheic alopecia,alopecia pityroides, and senile alopecia are not widely recognized andthere are hardly any therapies. With regard to cancer chemotherapydrug-induced alopecia, since body hair is rapidly lost over the wholebody, and this state continues for a long period, a large shock is givento the patient, but there are hardly any therapies therefor, and thereis a strong desire for the development of a therapy for cancertreatment. The same applies to alopecia due to radiation exposure as forcancer chemotherapy drug-induced alopecia.

Therefore, it is an object of the present invention to provide a novelagent for the treatment of alopecia for patients with alopecia, inparticular alopecia areata, androgenetic alopecia, postpartum alopecia,female pattern alopecia, seborrheic alopecia, alopecia pityroides,senile alopecia, cancer chemotherapy drug-induced alopecia, and alopeciadue to radiation exposure, the agent being not only effective and safebut also free from side effects such as an itching sensation,irritation, or feminization, not being contraindicated, and there beingno recurrence when the use thereof is stopped.

Furthermore, it is an object of the present invention to provide a newagent for the treatment and/or prevention of alopecia, the agent havinghair growth, hair restoration, and hair thickening effects, a white hairtherapeutic effect, and a terminal hair growth effect, regardless of thecause of the alopecia, that is, any cause such as male hormone, femalehormone, the balance thereof, any immune abnormality, inflammation,aging, or cancer chemotherapy drug, and to provide an agent for thetreatment and/or prevention of dandruff, white hair, and seborrheicscalp.

Means for Solving the Problems

As a result of an intensive investigation by the present inventors inlight of the above-mentioned circumstances, it has been found that anA-type natriuretic peptide (ANP) known as an acute heart failuretreatment drug, a B-type natriuretic peptide (BNP) known as a congestiveheart failure treatment drug, and a C-type natriuretic peptide (CNP)conventionally known as a vascular smooth muscle growth inhibitor, etc.have excellent effectiveness and safety as agents for the treatmentand/or prevention of alopecia, dandruff, white hair, and seborrheicscalp.

The agent for the treatment of alopecia of the present invention hasexcellent effectiveness and safety for in particular alopecia areata,androgenetic alopecia, postpartum alopecia, female pattern alopecia,seborrheic alopecia, alopecia pityroides, senile alopecia, cancerchemotherapy drug-induced alopecia, and alopecia due to radiationexposure. Moreover, the agent for the treatment of alopecia of thepresent invention exhibits a clear therapeutic effect toward alopeciahaving resistance to treatment with a steroid agent, an antihistaminedrug, Glycyron (registered trademark), carpronium chloride,cepharanthin, minoxidil, finasteride, cyclosporin A,Keishikaryukotsuboreito, Hangekobokuto, biotin, Anthralin, localimmunotherapy, cooling therapy, linearly polarized near-infrared rayirradiation therapy, or PUVA therapy, which are conventional treatmentmethods, and does not have side effects such as an itching sensation,irritation, or feminization, and it has been confirmed that even if theuse thereof is stopped there is no immediate recurrence, thus completingthe present invention.

Furthermore, in multilayer application such as with a combination of CNPor BNP with betamethasone valerate and gentamicin sulfate, a combinationof CNP or BNP with clobetasol propionate, a combination of CNP withcarpronium chloride, or a combination of CNP or BNP with minoxidil, ithas been confirmed that black terminal hair grows for alopecia otherthan alopecia areata, that is, androgenetic alopecia, postpartumalopecia, female pattern alopecia, seborrheic alopecia, alopeciapityroides, senile alopecia, cancer chemotherapy drug-induced alopecia,and alopecia due to radiation exposure, thus completing the presentinvention.

The present invention is specifically as follows.

[1] An agent for the treatment or prevention of alopecia, dandruff,itching, white hair, and seborrheic scalp, or a hair growth agent, ahair restoration agent, a hair loss progression preventive agent, a hairthinning progression preventive agent, a hair development promotionagent, a hair growth promotion agent, a hair cultivating agent, or anagent for the treatment or prevention of hair loss after childbirth orafter a disease, the agent containing a natriuretic peptide (NP) as anactive ingredient.[2] The treatment or prevention agent according to [1], wherein thealopecia is alopecia areata, androgenetic alopecia, postpartum alopecia,female pattern alopecia, seborrheic alopecia, alopecia pityroides,trichotillomania, cancer chemotherapy drug-induced alopecia, senilealopecia, or drug-induced alopecia.[3] The treatment or prevention agent according to [1], wherein thenatriuretic peptide (NP) is any of a CNP derivative, a BNP derivative,and an ANP derivative.[4] The treatment or prevention agent according to [1], wherein thenatriuretic peptide (NP) is any of a C-type natriuretic peptide (CNP), aB-type natriuretic peptide (BNP), and an A-type natriuretic peptide(ANP).[5] The treatment or prevention agent according to [1], wherein thenatriuretic peptide (NP) is any of CNP-22, CNP-53, and a CNP derivativehaving CNP activity in which any amino acid in the amino acid sequenceof CNP-22 or CNP-53 has been deleted, substituted, or added.[6] The treatment or prevention agent according to [1], wherein thenatriuretic peptide (NP) is CNP-22.[7] The treatment or prevention agent according to [1], wherein thenatriuretic peptide (NP) is any of BNP-26, BNP-32, BNP-45, and a BNPderivative having BNP activity in which any amino acid in the amino acidsequence of BNP-26, BNP-32, or BNP-45 has been deleted, substituted, oradded.[8] The treatment or prevention agent according to [1], wherein thenatriuretic peptide (NP) is BNP-32.[9] The treatment or prevention agent according to [1], wherein thenatriuretic peptide (NP) is an A-type natriuretic peptide (ANP), ANP28,or a BNP derivative having BNP activity in which any amino acid in theANP28 amino acid sequence has been deleted, substituted, or added.[10] The treatment or prevention agent according to [1], wherein thenatriuretic peptide (NP) is a chimeric peptide of two or morenatriuretic peptides (NPs) selected from ANP, BNP, and CNP, the chimericpeptide forming a cyclic structure by an intramolecular disulfide bond,

the CNP is a peptide selected from the group consisting of CNP-22,CNP-53, a peptide containing any amino acid sequence having 5 or morecontiguous amino acids from the CNP-22 amino acid sequence in which any1 to 5 amino acids have been deleted, substituted, or added, and apeptide containing any amino acid sequence having 5 or more contiguousamino acids from the CNP-53 amino acid sequence in which any 1 to 5amino acids have been deleted, substituted, or added,

the BNP is a peptide selected from the group consisting of BNP-26,BNP-32, BNP-45, a peptide containing any amino acid sequence having 5 ormore contiguous amino acids from the BNP-26 amino acid sequence in whichany 1 to 5 amino acids have been deleted, substituted, or added, apeptide containing any amino acid sequence having 5 or more contiguousamino acids from the BNP-32 amino acid sequence in which any 1 to 5amino acids have been deleted, substituted, or added, and a peptidecontaining any amino acid sequence having 5 or more contiguous aminoacids from the BNP-45 amino acid sequence in which any 1 to 5 aminoacids have been deleted, substituted, or added,

the ANP is ANP or a peptide containing any amino acid sequence having 5or more contiguous amino acids from the ANP amino acid sequence in whichany 1 to 5 amino acids have been deleted, substituted, or added, and

said chimeric peptide has CNP activity, BNP activity, or ANP activity,or the natriuretic peptide (NP) is a derivative of the chimeric peptideof NPs.

[11] The treatment or prevention agent according to [1], wherein itfurther comprises at least one medicinal agent selected from the groupconsisting of a steroid drug, an antihistamine drug, a vasodilator, amale hormone activity inhibitor, a female hormone drug, an antibiotic,an antifungal agent, pentadecane, cytopurine (6-benzylaminopurine),t-flavanone, adenosine, cepharanthin, Glycyron (registered trademark),which is a complex of glycyrrhizin, methionine, and glycine, cyclosporinA, Keishikaryukotsuboreito, Hangekobokuto, biotin, Anthralin,tacrolimus, and a tricyclic antidepressant.[12] The treatment or prevention agent according to [1], wherein itfurther contains at least one steroid drug selected from the groupconsisting of diflorasone, betamethasone, dexamethasone, clobetasol,prednisolone, mometasone, methylprednisolone, Deprodone, difluprednate,fluocinonide, amcinonide, triamcinolone, difluprednate, andhydrocortisone.[13] The treatment or prevention agent according to [1], wherein itfurther contains at least one antihistamine drug selected from the groupconsisting of azelastine, oxatomide, fexofenadine, emedastine, ebastine,cetirizine, bepotastine, olopatadine, and loratadine.[14] The treatment or prevention agent according to [1], wherein itfurther contains at least one vasodilator selected from the groupconsisting of minoxidil and carpronium chloride.[15] The treatment or prevention agent according to [1], wherein itfurther contains finasteride.[16] The treatment or prevention agent according to [1], wherein itfurther contains at least one female hormone drug selected from thegroup consisting of estrogen, estradiol, and progesterone.[17] The treatment or prevention agent according to [1], wherein itfurther contains at least one antibiotic or antifungal agent selectedfrom the group consisting of gentamicin, amphotericin B, nystatin,ketoconazole, terbinafine, flucytosine, fluconazole, itoraconazole,griseofulvin, and micafungin.[18] The treatment or prevention agent according to [1], wherein itfurther contains at least one medicinal agent selected from the groupconsisting of betamethasone, clobetasol, gentamicin, carproniumchloride, and minoxidil.[19] The treatment or prevention agent according to [1], wherein itrecovers hair growth with original hair color from a state in which hairhas grown with a paler color than the original hair color accompanyingalopecia.[20] The treatment or prevention agent according to [1], wherein itgrows black hair.[21] The treatment or prevention agent according to [1], wherein itchanges the hair quality to terminal hair.[22] The treatment or prevention agent according to [1], wherein itsuppresses the occurrence of dandruff or the scalp becoming seborrheic.[23] The treatment or prevention agent according to [2], wherein theandrogenetic alopecia is androgenetic alopecia in a male or androgeneticalopecia in a female.[24] The treatment or prevention agent according to [2], wherein theandrogenetic alopecia is androgenetic alopecia coexisting withseborrheic alopecia or alopecia pityroides.[25] The treatment or prevention agent according to [2], wherein thefemale pattern alopecia is female pattern alopecia coexisting withseborrheic alopecia or alopecia pityroides.[26] The treatment or prevention agent according to [1] or [23], whereinthe hair loss site is the frontal region or the crown.[27] The treatment or prevention agent according to [2], wherein theandrogenetic alopecia is type Va, VI, or VII on the Hamilton-Norwoodscale.[28] The treatment or prevention agent according to [1], wherein thealopecia areata is normal alopecia areata, alopecia totalis, alopeciauniversalis, or alopecia ophiasis.[29] The treatment or prevention agent according to [1], wherein thealopecia is alopecia of a target having a history of, or coexisting,allergic disease or autoimmune disease.[30] The treatment or prevention agent according to [29], wherein theautoimmune disease is any of chronic discoid lupus erythematosus,localized scleroderma, pemphigus, pemphigoid, herpes gestationis, linearIgA bullous dermatosis, acquired epidermolysis bullosa, vitiligo, orSutton's nevus.[31] The treatment or prevention agent according to [1], wherein thealopecia is alopecia of a target having a history of, or coexisting,allergic disease.[32] The treatment or prevention agent according to [1], wherein thealopecia is alopecia of a target having a history of, or coexisting,atopic disease.[33] The treatment or prevention agent according to [1], wherein thealopecia is alopecia of a target having a history of, or coexisting,autoimmune thyroid disease, vitiligo, systemic lupus erythematosus,rheumatoid arthritis, or myasthenia gravis.[34] The treatment or prevention agent according to [1], wherein thealopecia is alopecia accompanied by erythema, scale, or crust.[35] The treatment or prevention agent according to [1], wherein thealopecia is alopecia of a target having a history of, or coexisting,atopic dermatitis or allergic rhinitis.[36] The treatment or prevention agent according to [1], wherein thealopecia is alopecia of a target having a filaggrin gene abnormality andhaving a history of, or coexisting, atopic dermatitis.[37] The treatment or prevention agent according to [1], wherein thealopecia is alopecia of a target showing an allergic reaction toward atleast one allergen selected from the group consisting of house dust,mite, cedar, Dactylis, ragweed, and cat fur.[38] The treatment or prevention agent according to [1], wherein thealopecia is alopecia of a target showing an allergic reaction toward atleast one allergen selected from the group consisting of house dust,mite, cedar, Dactylis, ragweed, and cat fur in an allergen test by anyof a scratch test, an intradermal test, a patch test, and a specific IgEantibody in vitro assay.[39] The treatment or prevention agent according to [1] or [2], whereinthe alopecia is alopecia exhibiting resistance to treatment with asteroid drug, an antihistamine drug, Glycyron (registered trademark),carpronium chloride, cepharanthin, minoxidil, finasteride, cyclosporinA, Keishikaryukotsuboreito, Hangekobokuto, biotin, Anthralin, localimmunotherapy, cooling therapy, linearly polarized near-infrared rayirradiation therapy, PUVA therapy, or tacrolimus.[40] The treatment or prevention agent according to [1], wherein thealopecia is alopecia exhibiting resistance to treatment with a steroidtreatment agent.[41] The treatment or prevention agent according to [1], wherein thealopecia is alopecia of a target that has attained a steroid-dependentstate.[42] The treatment or prevention agent according to [1], wherein thealopecia is alopecia of a target for which a steroid treatment agentcannot be used.[43] The treatment or prevention agent according to [1], wherein thealopecia is alopecia exhibiting resistance to treatment with carproniumchloride.[44] The treatment or prevention agent according to [1], wherein thealopecia is alopecia exhibiting resistance to treatment withcepharanthin.[45] The treatment or prevention agent according to [1], wherein thealopecia is alopecia of a target for which a 5α-reductase type 2inhibitor cannot be used.[46] The treatment or prevention agent according to [1], wherein thealopecia is alopecia exhibiting resistance to treatment with anantiallergy drug.[47] The treatment or prevention agent according to [1], wherein thealopecia is alopecia exhibiting resistance to treatment with anantihistamine drug.[48] The treatment or prevention agent according to [1], wherein thealopecia is alopecia exhibiting resistance to treatment by stimulationtherapy using liquid nitrogen.[49] The treatment or prevention agent according to [1], wherein thealopecia is alopecia exhibiting resistance to treatment with minoxidil.[50] The treatment or prevention agent according to [1], wherein thealopecia is alopecia exhibiting resistance to treatment with a5α-reductase type 2 inhibitor.[51] The treatment or prevention agent according to [50], wherein the5α-reductase type 2 inhibitor is finasteride.[52] The treatment or prevention agent according to [1], wherein thealopecia is alopecia exhibiting resistance to treatment with anantifungal agent.[53] The treatment or prevention agent according to [2], wherein thealopecia areata is S1 or B0 alopecia or above.[54] The treatment or prevention agent according to [2], wherein thealopecia areata is S2 alopecia or above.[55] The treatment or prevention agent according to [2], wherein thealopecia areata is S3 alopecia or above.[56] The treatment or prevention agent according to [2], wherein thealopecia areata is S4 alopecia or above.[57] The treatment or prevention agent according to [2], wherein thealopecia areata is S5 alopecia or above.[58] The treatment or prevention agent according to [2], wherein thealopecia areata is B0 alopecia.[59] The treatment or prevention agent according to [2], wherein thealopecia areata is B1 alopecia.[60] The treatment or prevention agent according to [2], wherein thealopecia areata is B2 alopecia.[61] The treatment or prevention agent according to [1], wherein atherapeutic effect is obtained by application for 3 weeks or longer.[62] The treatment or prevention agent according to [1], wherein atherapeutic effect is obtained by application for 2 weeks or longer.[63] The treatment or prevention agent according to [1], wherein atherapeutic effect is obtained by application for 1 week or longer.[64] The treatment or prevention agent according to [1], wherein thereis no recurrence for a period of 1 month or longer even when applicationis stopped.[65] The treatment or prevention agent according to [1], wherein thereis no recurrence for a period of 2 months or longer even whenapplication is stopped.[66] The treatment or prevention agent according to [1], wherein thereis no recurrence for a period of 6 months or longer even whenapplication is stopped.[67] The treatment or prevention agent according to [1], wherein thedosage form is an ointment, a gel, a cream, a lotion, a liquid, a wax, apowder, a spray, a gel spray, a foam, a shampoo, a treatment, a scalptreatment, or a tonic.[68] The treatment or prevention agent according to [1], wherein thedosage form is an ointment or a gel.[69] The treatment or prevention agent according to [1], wherein thecontent of the natriuretic peptide (NP) is 1 to 1000 μg/g.[70] The treatment or prevention agent according to [1], wherein thecontent of the natriuretic peptide (NP) is 10 to 500 μg/g.[71] The treatment or prevention agent according to [1], wherein thecontent of the natriuretic peptide (NP) is 20 to 300 μg/g.[72] The treatment or prevention agent according to [1], wherein thecontent of the natriuretic peptide (NP) is 30 to 200 μg/g.[73] The treatment or prevention agent according to [1], wherein thecontent of the natriuretic peptide (NP) is 50 to 100 μg/g.[74] The treatment or prevention agent according to [1], wherein thealopecia is alopecia due to an endocrine disorder such asdyspituitarism, hypothyroidism, or hyperthyroidism, or a metabolicdisorder such as a nutritional disorder, hypoalbuminemia, cachexia,iron-deficiency anemia, zinc deficiency, homocystinuria, or cirrhosis ofthe liver, is telogenic alopecia due to high fever, childbirth, majorsurgery, etc., or is drug-induced alopecia due to an antithyroid drug,an anticoagulant, an antitumor drug, thallium, a psychotropic drug, aβ-blocker, etc.

Effects of the Invention

As is clear from the case tests described below, the treatment agent ofthe present invention containing a natriuretic peptide (NP) as an activeingredient can outstandingly improve alopecia areata, androgeneticalopecia, female pattern alopecia, seborrheic alopecia, alopeciapityroides, postpartum alopecia, senile alopecia, and cancerchemotherapy drug-induced alopecia. Furthermore, the treatment agent ofthe present invention can restore white hair to black hair or itsoriginal color. Moreover, in accordance with use of the treatment agentof the present invention, dandruff is decreased. Furthermore, thetreatment agent of the present invention does not have side effects suchas an itching sensation, irritation, and feminization, and there is norecurrence of alopecia areata and cancer chemotherapy drug-inducedalopecia for at least half a year even if its use is stopped.

The treatment agent of the present invention can be anticipated to beuseful as a very effective treatment drug for androgenetic alopecia, forwhich sufficient therapeutic effects cannot be obtained by theconventional minoxidil or finasteride, and alopecia areata, for whichthere are hardly any effective treatment methods. Furthermore, thetreatment agent of the present invention has marked hair growth, hairrestoration, and hair thickening effects for female pattern alopecia,seborrheic alopecia, alopecia pityroides, postpartum alopecia, senilealopecia, cancer chemotherapy drug-induced alopecia, and alopecia due toradiation exposure, for which there are hardly any therapies, candramatically decrease the amount of hair falling out, and can preventthe progress of hair loss.

Moreover, the treatment agent of the present invention can convertminiaturized hair root into large hair root that grows terminal hair andcan change the hair quality so that it is harder and thicker.Furthermore, the treatment agent of the present invention promotes hairgrowth of terminal hair, prolongs the growth phase, and increases longhair. Moreover, the treatment agent of the present invention promoteshair restoration and hair lengthening and speeds up the hair lengtheningrate.

Furthermore, the treatment agent of the present invention can increasethe number of hairs per hair follicle. The treatment agent of thepresent invention promotes hair growth and hair restoration in thefrontal region or M-shaped site, which is intractable, and has a hairgrowth effect, hair restoration effect, and hair thickening effect foralopecia that is classified as Va, VI, or VII on the Hamilton-Norwoodscale, which is wide area, severe androgenetic alopecia. The treatmentagent of the present invention restores hair for alopecia areata and hasthe effect of preventing restored hair from falling out such that newlygrown hair does not fall out after its application is stopped.

The treatment agent of the present invention can in particulardramatically improve severe alopecia on the adult head that has beendifficult to treat and causes problems in social life, without any sideeffects at all. The treatment agent of the present invention is not onlyeffective for intractable alopecia but also exhibits the same effectsfor both males and females and for both adults and younger people.

In the case of minoxidil and finasteride, which are conventionally used,there is the serious problem that when their use is stopped, theseverity prior to use immediately returns, but in the present invention,this defect is not seen for alopecia areata, postpartum alopecia, cancerchemotherapy drug-induced alopecia, and alopecia due to radiationexposure. Furthermore, with regard to androgenetic alopecia, not onlyfor the crown, for which a hair thickening effect by finasteride wasconfirmed, but also for the frontal region and the front temporal regionthe same hair growth, hair restoration, and hair thickening effects asfor the crown are confirmed.

Moreover, there is an excellent effect in improving seborrheic andpityriatic state of the scalp and preventing dandruff from occurring.The target of a treatment plan using minoxidil and finasteride byEuropean and American specialists is only Hamilton-Norwood scale ClassesII-V (Non-Patent Document 24); no effect is expected forHamilton-Norwood scale Classes Va, VI, and VII, and a hair follicletransplant or a wig is recommended. However, in accordance with thepresent invention, improvement effects such as the amount of hairfalling out dramatically decreasing, a longer growth phase, increase inlong hair, terminal hair growth, hair thickening and darkening, hairrestoration, promotion of hair lengthening, faster lengthening rate,black hair thickening, hair growing, and improvement of white hair areobserved 1 week to 3 weeks after application even for Hamilton-Norwoodscale Classes Va, VI, and VII.

Furthermore, steroid local injection, which is conventionally used foralopecia areata, has the difficulty that skin atrophy occurs at thelocal injection site and a substantial number of times of injection andtotal amount injected are required when the hair loss has spread to awide area, and local immunotherapy for alopecia areata has the problemthat there is the adverse event that it is associated with systemiccontact alopecia, local lymphadenopathy, and mechanical urticaria.However, in the present invention this defect is not only not seen atall, but the excellent effects that hair is newly grown, the therapeuticeffect for alopecia is not lost for a long period after its use isstopped, and restored hair is prevented from falling out have beenconfirmed. Even when application is stopped the effects from thetreatment agent of the present invention are maintained as a good skinstate for over half a year with respect to alopecia areata and cancerchemotherapy drug-induced alopecia.

Furthermore, the treatment agent of the present invention containing asan active ingredient ANP, BNP, CNP, a derivative of these NPs, achimeric peptide of 2 or more NPs selected from these NPs, or aderivative of a chimeric peptide of these NPs exhibits therapeuticeffects toward alopecia areata, which shows resistance to treatment withsteroid drugs, cooling therapy, antiallergy drugs, carpronium chloride,and cepharanthin. In particular, the treatment agent of the presentinvention containing as an active ingredient BNP, CNP, a derivative ofthese NPs, a chimeric peptide of these NPs, or a derivative of achimeric peptide of these NPs is also effective for alopecia areata thathas a large hair loss area and is intractable, and alopecia ophiasis,which is said to have a poor prognosis, and also exhibits marked effectson female pattern alopecia, postpartum alopecia, seborrheic alopecia,alopecia pityroides, senile alopecia, cancer chemotherapy drug-inducedalopecia, and minoxidil treatment-resistant androgenetic alopecia.

Furthermore, when the treatment agent of the present invention containsCNP or BNP as an active ingredient, the amount of hair falling outdecreases dramatically about 3 days after application, hair grows withapplication once or twice a day for 1 week to 2 weeks, and in many casesthe skin becomes difficult to see after application for 4 weeks.Following this, the hair continues to grow without continuousapplication, it becomes terminal hair, and there is a cure. The effectof the treatment agent of the present invention containing CNP or BNP asan active ingredient is strong, and even for alopecia ophiasis typealopecia areata, which is very intractable, hair growth recoversmarkedly.

In particular, the treatment agent of the present invention containingCNP as an active ingredient can more reliably suppress inflammation in ahair loss site than when BNP is an active ingredient, and symptoms suchas erythema, scale, and crust can be improved in a short period.Furthermore, the treatment agent of the present invention containing CNPas an active ingredient has a stronger effect in decreasing the amountof hair falling out, suppressing itchiness, and giving earlier hairgrowth than when BNP is an active ingredient.

Furthermore, when the treatment agent of the present inventioncontaining CNP as an active ingredient is applied only for 1 week to 2weeks, the hair growth effect is sustained for a longer period after theapplication is stopped, and hair continues to thicken. Therefore, wheninflammatory symptoms such as erythema or scale are seen in the hairloss site on the scalp, CNP is most recommended. This is a surprisingfinding even for the present inventors, who are dermatologists.Furthermore, when the treatment agent of the present invention containsANP as an active ingredient, it is effective only for alopecia in whichthere is no erythema, scale, seborrheic desquamation, etc. on the scalpand for which erythema, scale, seborrheic desquamation, etc. on thescalp that are induced by ANP application do not occur. In this case,hair grows with application twice a day for 1 week to 2 weeks.

Furthermore, the treatment agent of the present invention can suppressinflammation of seborrheic alopecia and alopecia pityroides and makehair grow. The treatment agent of the present invention can also promoterestoration of hair, dramatically decrease the amount of hair fallingout, make terminal hair grow, make hair thick and dark, restore hair,promote lengthening of hair, give a faster lengthening rate, andincrease long hair for female pattern alopecia, postpartum alopecia,senile alopecia, cancer chemotherapy drug-induced alopecia, and alopeciadue to radiation exposure.

Since the treatment agent of the present invention suppress skininflammation, it can make hair grow on a hair loss site of a targethaving a history of, or coexisting, autoimmune disease accompanied byskin symptoms such as chronic discoid lupus erythematosus, localizedscleroderma, pemphigus, pemphigoid, herpes gestationis, linear IgAbullous dermatosis, acquired epidermolysis bullosa, vitiligo, orSutton's nevus.

It is known that alopecia areata might be comorbid with an autoimmunedisease, and the treatment agent of the present invention can make hairgrow in a hair loss site of a target having a history of, or coexisting,autoimmune disease such as autoimmune thyroid disease, vitiligo,systemic lupus erythematosus, rheumatoid arthritis, or myastheniagravis.

Furthermore, since the treatment agent of the present inventionsuppresses inflammation and improves the state of scalp skin, it is alsoeffective for alopecia due to an endocrine disorder such asdyspituitarism, hypothyroidism, or hyperthyroidism or a metabolicdisorder such as a nutritional disorder, hypoalbuminemia, cachexia,iron-deficiency anemia, zinc deficiency, homocystinuria, or cirrhosis ofthe liver, telogenic alopecia due to high fever, childbirth, or majorsurgery, and alopecia medicamentosa due to an antithyroid drug, ananticoagulant, an antitumor drug, thallium, a psychotropic drug, or aβ-blocker.

In addition, an NPR-C receptor, which is common to NPs, is though to bea clearance receptor that does not mediate pharmacological action. It issaid that ANP and BNP bring about vasodilatory action, diuretic action,and cytostatic action by activation of an NPR-A receptor, it is thoughtthat CNP brings about vascular smooth muscle cell growth-inhibition byactivation of an NPR-B receptor, and it is therefore expected that theaction of CNP is different from that of BNP and ANP.

However, the present inventors actually carried out testing on patientswith alopecia, in particular patients with androgenetic alopecia andalopecia areata, and surprisingly it has been found that the treatmentagent of the present invention containing CNP or BNP as an activeingredient clearly shows higher therapeutic effects than when ANP is anactive ingredient, and among them the therapeutic effect was even higherwhen CNP was an active ingredient. On the other hand, the treatmentagent of the present invention containing BNP as an active ingredient ischaracterized in that black terminal hair often grows.

Natriuretic peptides (NPs), which are the active ingredients of thepresent invention, are hormones originally present in the body; thereare no concerns about side effects when applied to the skin, it isthought that there is only a slight effect on hemodynamics as long asthe dosage is appropriate, they can be used safely for a patient havinglow or unstable blood pressure, and it is therefore possible toadminister them for a long period to a patient with chronic alopecia.

Furthermore, they are effective for a patient exhibiting resistance totreatment with a steroid drug, carpronium chloride, an antifungal agent,cepharanthin, or minoxidil, hair grows with application twice a day for3 days to 2 weeks and, moreover, in the case of alopecia areata andcancer chemotherapy drug-induced alopecia, after administration isstopped alopecia symptoms do not recur at the application site. Inaddition, the treatment agent of the present invention is anunprecedented and outstanding treatment agent having the advantage thatit can be used for children and females.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 A diagram comparing the amino acid sequence of human CNP peptide,the amino acid sequence of human BNP peptide, and the amino acidsequence of human ANP peptide. Each character denotes the type of aminoacid as a one letter code. The line connecting C (cysteine) and C(cysteine) denotes an intramolecular disulfide bond. The amino acidsequence of human CNP peptide and the amino acid sequences of human BNPpeptide and human ANP peptide have three common regions, that is, anamino acid sequence represented by ‘CFG’, an amino acid sequencerepresented by ‘DRI’, and an amino acid sequence represented by ‘SGLGC’,and four mutually different sequences separated by these three commonsequences.

FIG. 2 A diagram showing the therapeutic effects on alopecia areata froman ANP gel. The severity of the alopecia areata was evaluated in termsof the proportion of bald patch area relative to the entire head inaccordance with the USA alopecia areata evaluation guidelines, and acomparison was made for each case between that before application of theANP gel and that after the application thereof. Each point on theleft-hand side denotes the severity before application and each point onthe right-hand side denotes the severity after application. The pointfor the severity before application and the point for the severity afterapplication from the same test subject are connected by a straight line.The ANP gel rarely exhibited a fixed effect in the treatment of alopeciaareata in those cases where no symptoms of irritation occurred, but inmany cases there was no change in the proportion of the bald patch area,and itchiness occurred in many cases. Furthermore, the ANP gel ratheraggravated erythema, scale, itchiness, and dandruff and increased theamount of hair falling out, and its use was stopped in most cases; thistendency was particularly marked for androgenetic alopecia.

FIG. 3 A diagram showing the therapeutic effects on alopecia areata froma BNP gel. The severity of alopecia areata was evaluated in terms of theproportion of bald patch area relative to the entire head in accordancewith the USA alopecia areata evaluation guidelines, and a comparison wasmade for each case between that before application of the BNP gel andthat after application thereof. Each point on the left-hand side denotesthe severity before application and each point on the right-hand sidedenotes the severity after application. The point for the severitybefore application and the point for the severity after application fromthe same test subject are connected by a straight line. It was foundthat the BNP gel was effective in the treatment of alopecia areata inall cases.

FIG. 4 A diagram showing the therapeutic effects on alopecia areata froma BNP ointment. The severity of alopecia areata was evaluated in termsof the proportion of bald patch area relative to the entire head inaccordance with the USA alopecia areata evaluation guidelines, and acomparison was made for each case between that before application of theBNP ointment and that after application thereof. Each point on theleft-hand side denotes the severity before application and each point onthe right-hand side denotes the severity after application. The pointfor the severity before application and the point for the severity afterapplication from the same test subject are connected by a straight line.It was found that the BNP ointment was effective in the treatment ofalopecia areata in all cases.

FIG. 5 A diagram showing the therapeutic effects on alopecia areata froma CNP gel. The severity of alopecia areata was evaluated in terms of theproportion of bald patch area relative to the entire head in accordancewith the USA alopecia areata evaluation guidelines, and a comparison wasmade for each case between that before application of the CNP gel andthat after application thereof. Each point on the left-hand side denotesthe severity before application and each point on the right-hand sidedenotes the severity after application. The point for the severitybefore application and the point for the severity after application fromthe same test subject are connected by a straight line. It was foundthat the CNP gel was effective in the treatment of alopecia areata. Incase C1, because it was the 7^(th) day of CNP gel application thepresence or absence of a therapeutic effect could not be confirmedclearly.

FIG. 6 A diagram showing the therapeutic effects on alopecia areata froma CNP ointment. The severity of alopecia areata was evaluated in termsof the proportion of bald patch area relative to the entire head inaccordance with the USA alopecia areata evaluation guidelines, and acomparison was made for each case between that before application of theCNP ointment and that after application thereof. Each point on theleft-hand side denotes the severity before application and each point onthe right-hand side denotes the severity after application. The pointfor the severity before application and the point for the severity afterapplication from the same test subject are connected by a straight line.It was found that the CNP ointment exhibited clear effects in thetreatment of alopecia areata except in one case, that of case C14. Incase C14, insofar as the bald patch area could be evaluated there was nohair growth that covered the entire bald patch, but clear growth ofterminal hair was confirmed.

FIG. 7 A photographic diagram showing the therapeutic effect when an ANPgel was applied to the back of the head of alopecia areata case A2 testsubject. P denotes a bald patch before application and T denotes thebald patch of the same site after 100 μg/g ANP gel was applied twice aday for 5 weeks. Clear hair growth was observed.

FIG. 8-1 A photographic diagram showing the therapeutic effect when anANP gel was applied to the right temporal region of alopecia areata caseA3 test subject (case C15 test subject). The test subject faced towardthe right in the photograph. P denotes a bald patch before applicationand T1 denotes the bald patch of the same site after 100 μg/g CNPointment was applied twice a day for 2 weeks and from the next day afterthat 100 μg/g ANP gel was applied twice a day for 2 weeks. Strong hairgrowth was observed.

FIG. 8-2 A photographic diagram showing the therapeutic effect when anANP gel was applied to the right temporal region of alopecia areata caseA3 test subject (case C15 test subject). The test subject faced towardthe right in the photograph. T2 denotes the bald patch of the same siteafter 100 μg/g CNP ointment was applied twice a day for 2 weeks and fromthe next day after that 100 μg/g ANP gel was applied twice a day for 5weeks. Compared with that before application and that of T1 in FIG. 8-1,stronger hair growth was observed.

FIG. 9 A photographic diagram showing the therapeutic effect when an ANPgel was applied to alopecia areata case A4 test subject. P denotes abald patch before application and T denotes the bald patch of the samesite after 100 μg/g ANP gel was applied twice a day for 4 weeks. It canbe seen that the bald patch was cured.

FIG. 10 A photographic diagram showing the therapeutic effect when anANP gel and a BNP gel were applied to alopecia areata case A5 testsubject (case B3 test subject). The test subject faced toward the rightin the photograph. P denotes a bald patch before application, T1 denotesthe bald patch of the same site after 100 μg/g ANP gel was applied twicea day for 3 weeks, and T2 denotes the bald patch of the same site afterapplication of the ANP gel was stopped at 3 weeks and from the next dayafter that 100 μg/g BNP gel was applied twice a day for 2 weeks.Although the ANP gel enlarged the hair loss range somewhat, marked hairgrowth was observed with the BNP gel.

FIG. 11 A photographic diagram showing the therapeutic effect when anANP gel was applied to the crown of alopecia areata case A6 test subject(case C3 and C4 test subject). The test subject faced upward in thephotograph. P denotes a bald patch before application, and T denotes thebald patch of the same site after 100 μg/g ANP gel was applied twice aday for 2 weeks. In the case of this test subject, there was no effect,and the hair loss range was somewhat enlarged.

FIG. 12-1 A photographic diagram showing the therapeutic effect when aBNP gel was applied to the right temporal region of alopecia areata caseB1 test subject (case C12 test subject). The test subject faced towardthe right in the photograph. P denotes a bald patch before applicationand T1 denotes the bald patch of the same site after 100 μg/g BNP gelwas applied twice a day for 7 days. Clear hair growth was observed.

FIG. 12-2 A photographic diagram showing the therapeutic effect when aBNP gel was applied to the right temporal region of alopecia areata caseB1 test subject (case C12 test subject). The test subject faced towardthe right in the photograph. T2 denotes the bald patch of the same siteafter 100 μg/g BNP gel was applied twice a day for 23 days, and T3denotes the bald patch of the same site after application of 100 μg/gBNP gel was stopped at 24 days and 5 months had elapsed. Hair growth wasobserved to such an extent that the location of the bald patch wassubstantially lost, and the hair growth continued after application wasstopped.

FIG. 13 A photographic diagram showing the therapeutic effect when a BNPgel was applied to alopecia areata case B2 test subject. P denotes abald patch before application and T1 denotes the bald patch of the samesite after 50 μg/g BNP gel was applied twice a day for 7 days. Clearhair growth was observed and there was a cure.

FIG. 14 A photographic diagram showing the therapeutic effect when a BNPgel was applied to the left frontal region of alopecia areata case B4test subject (test subject of test subject C9). The test subject faceddownward in the photograph. P denotes a bald patch before application,T1 denotes the bald patch of the same site after 100 μg/g BNP gel wasapplied twice a day for 2 weeks, T2 denotes the bald patch of the samesite after 100 μg/g BNP gel was applied twice a day for 3 weeks, and T3denotes the bald patch of the same site after application of 100 μg/gBNP gel was stopped at 3 weeks and 3 weeks had elapsed. Marked hairgrowth and hair thickening were observed.

FIG. 15-1 A photographic diagram showing the therapeutic effect when aBNP ointment was applied to a bald area of the crown of alopecia areatacase B5 test subject. The test subject faced upward in the photograph. Pdenotes the bald patch before application, and T1 denotes the bald patchof the same site after 50 μg/g BNP ointment was applied twice a day for2 weeks and from the next day after that 30 μg/g BNP ointment wasapplied twice a day for 1 week. Clear hair growth was observed.

FIG. 15-2 A photographic diagram showing the therapeutic effect when aBNP ointment was applied to the bald area of the crown of alopeciaareata case B5 test subject. The test subject faced upward in thephotograph. T2 denotes the bald patch of the same site after 50 μg/g BNPointment was applied twice a day for 2 weeks, from the next day afterthat 30 μg/g BNP ointment was applied twice a day for 1 week, and afterthat a further 50 μg/g BNP ointment was applied twice a day for 2 weeks.Compared with T1 in FIG. 15-1, further marked hair growth and hairthickening were observed.

FIG. 16 A photographic diagram showing the therapeutic effect when a BNPointment was applied to a bald area of the right temporal region ofalopecia areata case B5 test subject. The test subject faced toward theright in the photograph. P denotes the bald patch before application,and T1 denotes the bald patch of the same site after 50 μg/g BNPointment was applied twice a day for 2 weeks, from the next day afterthat 30 μg/g BNP ointment was applied twice a day for 1 week, and afterthat a further 50 μg/g BNP ointment was applied twice a day for 2 weeks.Marked hair growth that covered the entire bald patch was observed.

FIG. 17 A photographic diagram showing the therapeutic effect when a CNPgel was applied to the crown of alopecia areata case C1 test subject. Pdenotes a bald patch before application, and T denotes the bald patch ofthe same site after 100 μg/g CNP gel was applied twice a day for 1 week.Clear hair growth was observed.

FIG. 18-1 A photographic diagram showing the therapeutic effect when aCNP ointment was applied to a bald patch of the crown of alopecia areatacase C2 test subject (case C11 test subject). P denotes a bald patchbefore application, and T1 denotes the bald patch of the same site after100 μg/g CNP ointment was applied twice a day for 1 week. Clear hairgrowth was observed.

FIG. 18-2 A photographic diagram showing the therapeutic effect when aCNP ointment and a CNP gel were applied to the bald patch of the crownof alopecia areata case C2 test subject (case C11 test subject). T2denotes the bald patch of the same site after 100 μg/g CNP ointment wasapplied twice a day for 1 week and from the next day after that 100 μg/gCNP gel was applied twice a day for 1 week, and T3 denotes the baldpatch of the same site after 100 μg/g CNP ointment was applied twice aday for 1 week and from the next day after that 100 μg/g CNP gel wasapplied twice a day for 3 weeks. Clear hair growth and enlargement ofthe hair growth region were observed.

FIG. 19-1 A photographic diagram showing the therapeutic effect when aCNP ointment was applied to a bald patch of the frontal region ofalopecia areata case C2 test subject (case C11 test subject). The testsubject faced downward in the photograph. P denotes the bald patchbefore application, and T1 denotes the bald patch of the same site after100 μg/g CNP ointment was applied twice a day for 1 week. Clear hairgrowth was observed.

FIG. 19-2 A photographic diagram showing the therapeutic effect when aCNP ointment and a CNP gel were applied to the bald patch of the frontalregion of alopecia areata case C2 test subject (case C11 test subject).The test subject faced downward in the photograph. T2 denotes the baldpatch of the same site after 100 μg/g CNP ointment was applied twice aday for 1 week and from the next day after that 100 μg/g CNP gel wasapplied twice a day for 3 weeks. Compared with T1 in FIG. 19-1, clearhair growth and enlargement of the hair growth region were observed.

FIG. 20 A photographic diagram showing the therapeutic effect when a CNPgel was applied to a bald patch of the left temporal region of alopeciaareata case C3 test subject (case A6 and C4 test subject). The testsubject faced toward the left in the photograph. P denotes the baldpatch before application, T1 denotes the bald patch of the same siteafter 100 μg/g CNP gel was applied twice a day for 2 weeks, and T2denotes the bald patch of the same site after 100 μg/g CNP gel wasapplied twice a day for 3 weeks and after that application of CNP gelwas stopped and 6 months had elapsed. Clear hair growth and enlargementof the hair growth region were observed.

FIG. 21 A photographic diagram showing the therapeutic effect when a CNPointment was applied to a bald patch of the left temporal region ofalopecia areata case C5 test subject (case A1 test subject). The testsubject faced downward in the photograph. P denotes the bald patchbefore application, T1 denotes the bald patch of the same site after 100μg/g CNP ointment was applied twice a day for 3 weeks, and T2 denotesthe bald patch of the same site after 100 μg/g CNP gel was applied twicea day for 3 weeks and after that application of CNP ointment was stoppedand 6 months had elapsed. Clear hair growth and enlargement of the hairgrowth region were observed.

FIG. 22 A photographic diagram showing the therapeutic effect when a CNPointment was applied to a bald patch of the right frontal region ofalopecia areata case C6 test subject (case A7 test subject). The testsubject faced toward the right in the photograph. P denotes the baldpatch before application, and T denotes the bald patch of the same siteafter 50 μg/g CNP ointment was applied twice a day for 3 weeks. Cleargrowth of terminal hair was observed.

FIG. 23 A photographic diagram showing the therapeutic effect when a CNPointment was applied to alopecia areata case C7 test subject. P denotesa bald patch before application, and T denotes the bald patch of thesame site after 100 μg/g CNP ointment was applied twice a day for 2weeks. Clear hair growth was observed.

FIG. 24-1 A photographic diagram showing the therapeutic effect when aCNP ointment was applied to alopecia areata case C10 test subject (caseB6 test subject). P denotes a bald patch before application, and T1denotes the bald patch of the same site after 100 μg/g CNP ointment wasapplied twice a day for 2 weeks. Clear hair growth was observed.

FIG. 24-2 A photographic diagram showing the therapeutic effect when aCNP ointment was applied to alopecia areata case C10 test subject (caseB6 test subject). T2 denotes the bald patch of the same site after 100μg/g CNP ointment was applied twice a day for 3 weeks. Compared with T1in FIG. 24-1, clearer hair growth was observed.

FIG. 25 A photographic diagram showing the therapeutic effect when a CNPointment was applied to a bald patch of the left temporal region ofalopecia areata case C12 test subject (case B1 test subject). The testsubject faced toward the left in the photograph. P denotes the baldpatch before application, T1 denotes the bald patch of the same siteafter 100 μg/g CNP ointment was applied twice a day for 3 weeks, and T2denotes the bald patch of the same site after 100 μg/g CNP ointment wasapplied twice a day for 5 weeks. Enlargement of the hair growth regionover time and formation of terminal hair were clearly observed.

FIG. 26 A photographic diagram showing the therapeutic effect when a CNPointment was applied to alopecia areata case C13 test subject. P denotesa bald patch before application, and T denotes the bald patch of thesame site after 100 μg/g CNP ointment was applied twice a day for 2weeks. Clear hair growth was observed.

FIG. 27 A photographic diagram showing the therapeutic effect when anANP gel was applied to the crown of androgenetic alopecia case A8 testsubject (case B8 test subject). P denotes a bald patch beforeapplication, and T denotes the bald patch of the same site after 100μg/g ANP gel was applied twice a day for 2 weeks. Hair growth was notobserved at all.

FIG. 28-1 A photographic diagram showing the therapeutic effect when aBNP gel was applied to the crown of androgenetic alopecia case B7 testsubject. P denotes a bald patch before application, and T1 denotes thebald patch of the same site after 100 μg/g BNP gel was applied twice aday for 10 days. Hair growth and formation of terminal hair wereobserved.

FIG. 28-2 A photographic diagram showing the therapeutic effect when aBNP gel was applied to the crown of androgenetic alopecia case B7 testsubject. T2 denotes the bald patch of the same site after 100 μg/g BNPgel was applied twice a day for 14 days and from the next day after thatapplication of the BNP gel was stopped and 6 months had elapsed.Compared with T1 in FIG. 28-1, formation of terminal hair progressed,the area of thinning hair was reduced, and a substantially normal statewas recovered.

FIG. 29 A photographic diagram showing the therapeutic effect when a BNPgel was applied to the crown of androgenetic alopecia case B12 testsubject. P denotes a bald patch before application, T1 denotes the baldpatch of the same site after 50 μg/g BNP gel was applied twice a day for3 days, and T2 denotes the bald patch of the same site after 50 μg/g BNPgel was applied twice a day for 21 days. Clear hair growth and formationof terminal hair were observed.

FIG. 30-1 A photographic diagram showing the therapeutic effect when aCNP ointment was applied to the crown and frontal region of androgeneticalopecia case C16 test subject. The test subject faced downward in thephotograph. P denotes a bald patch before application, and T1 denotesthe bald patch of the same site after 100 μg/g CNP ointment was appliedtwice a day for 3 weeks. Clear hair growth and formation of terminalhair were observed.

FIG. 30-2 A photographic diagram showing the therapeutic effect when aCNP ointment was applied to the crown and frontal region of androgeneticalopecia case C16 test subject. The test subject faced downward in thephotograph. T2 denotes the bald patch of the same site after 100 μg/gCNP ointment was applied twice a day for 4 weeks. Compared with thatbefore application and that of T1 in FIG. 30-1, very marked hair growthover time and formation of terminal hair were observed.

FIG. 31 A photographic diagram showing the therapeutic effect when a CNPointment was applied to androgenetic alopecia case C17 test subject. Thetest subject faced downward in the photograph. P denotes a bald patchbefore application, and T denotes the bald patch of the same site after100 μg/g CNP ointment was applied twice a day for 1 week. Clear hairgrowth and formation of terminal hair were observed.

FIG. 32 A photographic diagram showing the therapeutic effect when a CNPgel was applied to postpartum alopecia case C18 test subject. The testsubject faced downward in the photograph. P denotes a bald patch beforeapplication, and T denotes the bald patch of the same site after 100μg/g CNP gel was applied twice a day for 1 week. Clear hair growth andformation of terminal hair were observed.

FIG. 33 A photographic diagram showing the therapeutic effect when a CNPgel was applied to female pattern alopecia case C19 test subject. Thetest subject faced downward in the photograph. P denotes a bald patchbefore application, and T denotes the bald patch of the same site after100 μg/g CNP gel was applied twice a day for 4 days. Although it wasshort, clear hair growth was observed.

FIG. 34 A photographic diagram showing the therapeutic effect when a BNPgel was applied to a circular bald area of the crown of alopecia areatacase B13 test subject (case C13 test subject). The test subject faceddownward in the photograph. P denotes the bald patch before application,and T denotes the bald patch of the same site after 50 μg/g BNP gel wasapplied twice a day for 2 weeks. There was no hair on the scalp of thebald area within the range encircled by the dotted line in P, whereasclear hair growth was observed on the scalp of the bald area within therange encircled by the dotted line in T.

FIG. 35 A photographic diagram showing the therapeutic effect when a CNPgel was applied to a circular bald area of the left temporal region ofalopecia areata case C20 test subject. The test subject faced downwardand toward the left in the photograph in P2, and upward and toward theleft in T2. P2 denotes a hair loss site before application, and T2denotes the same site after 50 μg/g CNP gel was applied twice a day for2 weeks. The areas photographed in photographs P2 and T2 weresubstantially identical to each other. There was almost no hair growthon the scalp of the bald area within the range encircled by the dottedline in P2, whereas a large amount of hair growth was observed on thescalp within the range encircled by the dotted line in T2.

FIG. 36 A photographic diagram showing the therapeutic effect when aBNP:betamethasone:gentamicin combination was applied to a circular baldarea of the crown of alopecia areata case B14 test subject. The testsubject faced upward in the photograph. P denotes the bald patch beforeapplication, and T denotes the bald patch of the same site after a 50μg/g BNP:600 μg/mL betamethasone:500 μg/mL gentamicin combination wasapplied twice a day for 7 days. There was no hair growth in a centralpart of the bald area within the range encircled by the dotted line inP, whereas a large amount of hair growth was observed in the middle ofthe bald area within the range encircled by the dotted line in T.

FIG. 37 A photographic diagram showing the therapeutic effect when aCNP: betamethasone: gentamicin combination was applied to a circularbald area of the right crown of alopecia areata case C21 test subject.The test subject faced rearward in the photograph of P and upward andtoward the left in the photograph of T. P denotes the bald patch beforeapplication, and T denotes the bald patch of the same site after a 50μg/g CNP:600 μg/mL betamethasone:500 μg/mL gentamicin combination wasapplied twice a day for 3 weeks and then stopped for 1 week. In P theskin of the scalp of the bald area within the range encircled by thedotted line could be seen and there was only slight growth of very finewhite downy hair, whereas in T black hair grew densely on the scalp ofthe bald area within the range encircled by the dotted line.

FIG. 38 A photographic diagram showing the therapeutic effect when aCNP:betamethasone:gentamicin combination was applied to a circular baldarea of the crown of alopecia areata case C22 test subject. The testsubject faced toward the left in the photograph. P denotes the baldpatch before application, and T denotes the bald patch after 50 μg/g CNPgel was applied twice a day for 2 weeks and subsequently a 50 μg/gCNP:600 μg/mL betamethasone:500 μg/mL gentamicin combination was appliedtwice a day for 2 weeks. In P there was no hair growth at all on thescalp of the bald area within the range encircled by the dotted line,whereas in T black hair grew densely on the scalp, at the upper andlower edges of the photograph, of the bald area within the rangeencircled by the dotted line.

FIG. 39 A photographic diagram showing the therapeutic effect when a CNPgel was applied to a circular bald area of the back of the head ofalopecia areata case C23 test subject. The test subject faced rearwardin the photograph. P denotes the bald patch before application, and Tdenotes the bald patch of the same site after 50 μg/g CNP gel wasapplied twice a day for 2 weeks. In P the hair growth in the bald areawithin the range encircled by the dotted line was scattered with finehair, whereas in T the hair growth in the bald area within the rangeencircled by the dotted line was dense with thick and sturdy hair.

FIG. 40 A photographic diagram showing the therapeutic effect when a BNPgel was applied to a thinning hair site of the frontal region and thecrown of androgenetic alopecia case B15 test subject. The test subjectfaced downward in the photograph. P denotes the thinning hair sitebefore application, and T denotes the hair loss site of the same siteafter 50 μg/g BNP gel was applied twice a day for 2 weeks. In P the hairgrowth of the hair loss site was scattered with fine hair, whereas in Tthe hair growth was dense with thick sturdy hair. In particular,focusing on the skin of the M-shaped site, it could be seen that thehair growth was dense.

FIG. 41 A photographic diagram showing the therapeutic effect when a BNPgel was applied to a hair loss site of the crown and the frontal regionof androgenetic alopecia case B16 test subject. The test subject faceddownward in the photograph. P denotes the hair loss site beforeapplication, T1 denotes the hair loss site of the same site after 100μg/g BNP gel was applied twice a day for 3 weeks, and T2 denotes thehair loss site of the same site when the application of 100 μg/g BNP gelwas stopped after 3 weeks and from the next day 200 μg/g BNP gel wasapplied twice a day for 2 weeks. In P hair was scattered in the hairloss site within the range encircled by the dotted line, whereas in T1hair with a feeling of volume grew densely in a central part within therange encircled by the dotted line, and in T2 the hair grew more denselyand the so-called M-shaped hair loss site disappeared. In the photographof T2, the reason why a central part within the range encircled by thedotted line, the so-called O-shaped hair loss site, was conspicuous isbecause the photograph was taken from a camera angle directly above thecrown such that the crown, which was the remaining hair loss site, couldbe seen well.

FIG. 42 A photographic diagram showing the therapeutic effect when a CNPgel was applied to a hair loss site of the crown of androgeneticalopecia case C24 test subject. The test subject faced downward in thephotograph. P denotes the bald patch before application, and T denotesthe bald patch of the same site after 50 μg/g CNP gel was applied twicea day for 1 week. In P hair was scattered and the scalp could be seen,whereas in T hair with a feeling of volume grew and it was difficult tosee the scalp.

FIG. 43 A photographic diagram showing the therapeutic effect when aCNP:betamethasone:gentamicin combination was applied to a hair loss siteof the frontal region of androgenetic alopecia case C25 test subject.The test subject faced forward in the photograph. P1 denotes the hairloss site before application, and T1 denotes the hair loss site of thesame site after a 25 μg/g CNP:600 μg/mL betamethasone:500 μg/mLgentamicin combination was applied twice a day for 12 days. Comparedwith the photograph of P1, in the photograph of T1 the feeling of volumeof the hair increased, and the scalp of the frontal region was moredifficult to see.

FIG. 44 A photographic diagram showing the therapeutic effect when aCNP:betamethasone:gentamicin combination was applied to a hair loss siteof the frontal region of androgenetic alopecia case C25 test subject.The test subject faced toward the right in the photograph, with theright frontal region visible. P2 denotes the hair loss site beforeapplication, and T2 denotes the hair loss site of the same site after a25 μg/g CNP:600 μg/mL betamethasone:500 μg/mL gentamicin combination wasapplied twice a day for 12 days. Compared with the photograph of P2, inthe photograph of T2 the feeling of volume of the hair increased, andthe scalp of the frontal region was more difficult to see.

FIG. 45 A photographic diagram showing the therapeutic effect when aCNP:clobetasol combination was applied to a hair loss site of thefrontal region and the crown of androgenetic alopecia case C26 testsubject. The test subject faced downward in the photograph. P denotesthe hair loss site before application, and T denotes the hair loss siteof the same site after a 50 μg/mL CNP:250 μg/g clobetasol combinationwas applied twice a day for 1 week. Compared with the photograph of P,in the photograph of T the density of the hair increased, each hairthickened, and even black hairs increased.

FIG. 46 A photographic diagram showing the therapeutic effect when a CNPgel was applied to a male pattern hair loss site at the M-shaped hairline of the right frontal region of androgenetic alopecia case C27 testsubject. The test subject faced toward the right in the photograph. Pdenotes the hair loss site before application, and T denotes the samesite after 50 μg/g CNP gel was applied twice a day for 2 weeks. In Pthere was hardly any hair on the scalp of the hair loss site, whereas inT a large amount of hair growth was observed on the scalp of the hairloss site.

FIG. 47 A photographic diagram showing the therapeutic effect when anANP gel or a BNP gel was applied to a hair loss site of the crown ofpostpartum alopecia case A10 (B17) test subject. The test subject faceddownward in the photograph. P denotes the hair loss site beforeapplication, T1 denotes the same site after 100 μg/g ANP gel was appliedtwice a day for 3 weeks, and T2 denotes the same site when applicationof the ANP gel was ended after 3 weeks and application was suspended for6 months, following which 50 μg/g BNP gel was applied to the hair losssite of the crown once every 3 to 4 days for 3 weeks. In photograph (T1)after application of the ANP gel, the hair loss range enlarged, but inphotograph (T2) after application of the BNP gel, thinning hair on thecrown thickened to such an extent that it was not noticeable to the eye.

FIG. 48 A photographic diagram showing the therapeutic effect when a CNPgel was applied to a hair loss site at the parting on the frontal regionof female pattern alopecia case B19 test subject. The test subject faceddownward in the photograph. P denotes the hair loss site beforeapplication, and T denotes the same site after 50 μg/g BNP gel wasapplied twice a day for 1 week. Comparing the two, in the photograph ofP the scalp had scattered hair growth, but in the photograph of T hairgrowth could be confirmed on the scalp.

FIG. 49 A photographic diagram showing the therapeutic effect when a BNPgel was applied to a thinning hair part of the crown of female patternalopecia case B20 test subject. The test subject faced downward in thephotograph. P denotes the thinning hair part before application, and Tdenotes the same site after 50 μg/g BNP gel was applied twice a day for2 weeks. After the BNP gel was applied for 2 weeks, the thinning hairbecame inconspicuous.

FIG. 50 A photographic diagram showing the therapeutic effect when a CNPgel was applied to a thinning hair part of the frontal region and thecrown of female pattern alopecia case C28 test subject. The test subjectfaced downward in the photograph. P denotes the thinning hair partbefore application, and T denotes the same site after 50 μg/g CNP gelwas applied once or twice a day for 4 weeks. After the CNP gel wasapplied for 4 weeks, the hair thickened to such an extent that thinninghair could hardly be seen.

FIG. 51 A photographic diagram showing the therapeutic effect when a BNPgel was applied to a thinning hair part of the frontal region and thecrown of case B23 test subject with coexisting senile alopecia andalopecia pityroides. The test subject faced downward in the photograph.P denotes the thinning hair part before application, and T denotes thesame site when 50 μg/g BNP gel was applied for 2 weeks, application wasthen suspended for 1 week, and 200 μg/g BNP gel was applied once a dayfor 5 days. After the BNP gel was applied, pityriatic desquamation wasalleviated, vellus hair turned into terminal hair and became black, andthe thinning hair was greatly improved.

FIG. 52 A photographic diagram showing the therapeutic effect when a CNPgel was applied to a thinning hair part of the frontal region and thecrown of alopecia pityroides case C31 test subject. The test subjectfaced downward in the photograph. P denotes the thinning hair partbefore application, and T denotes the same site after 100 μg/g CNP gelwas applied twice a day for 2 weeks. After the CNP gel was applied, aconsiderable amount of thickly adhering scale was lost, and hair startedto thicken on the crown.

FIG. 53 A photographic diagram showing the therapeutic effect when a BNPgel was applied to a thinning hair part of the frontal region and thecrown of alopecia pityroides case B28 test subject. The test subjectfaced downward in the photograph. P denotes the thinning hair partbefore application, and T denotes the same site after 20 μg/g BNP gelwas applied for 1 week. After the BNP gel was applied, thicklyaccumulated scale disappeared, and fairly clear hair growth and hairthickening were observed from the frontal region to the crown.

FIG. 54 A photographic diagram showing the therapeutic effect when anANP gel or a BNP gel was applied to a thinning hair part of the frontalregion and the crown of senile alopecia case A15 test subject. The testsubject faced downward in the photograph. P denotes the thinning hairpart before application, T1 denotes the thinning hair part after 100μg/g ANP gel was applied twice a day for 3 weeks, and T2 denotes thesame site when 50 μg/g CNP gel was applied twice a day for 4 weeks fromthe next day after the ANP gel had been applied for 1 week. After theCNP gel was applied, clear hair thickening was seen in the frontalregion, and hair stopped falling out.

FIG. 55 A photographic diagram showing the therapeutic effect when a BNPgel was applied to a bald area of the back of the head of alopeciaareata case B26 test subject. The test subject faced rearward in thephotograph. P denotes the bald area before application, T1 denotes thesame site after 50 μg/g BNP gel was applied twice a day for 2 weeks, andT2 denotes the same site when application of the BNP gel was ended after2 weeks and 6 weeks had elapsed after that. Marked hair growth was seenin the bald area to which the BNP gel had been applied.

FIG. 56-1 A photographic diagram showing the therapeutic effect when aCNP gel was applied to a bald area of the frontal region and the crownof cancer chemotherapy drug-induced alopecia case C35 test subject. Thetest subject faced downward in the photograph. P denotes the hair losssite before application, and T denotes the same site after 50 μg/gconcentration CNP gel was applied to an area from the frontal region tothe crown twice a day for 6 weeks. The number of hairs increased in thehair loss site to which the CNP gel had been applied, considerable hairthickening was observed, and it was confirmed that the hairs had becomethick and long.

FIG. 56-2 A photographic diagram showing the therapeutic effect when a50 μg/g CNP:600 μg/mL betamethasone:500 μg/mL gentamicin combination wasapplied to a circular bald area occurring in the left temporal region ofcancer chemotherapy drug-induced alopecia case C35 test subject. Thetest subject faced toward the left in the photograph. P denotes the hairloss site before application, and T denotes the same site after the 50μg/g CNP:600 μg/mL betamethasone:500 μg/mL gentamicin combination wasapplied twice a day for 3 weeks. Marked terminal hair growth wasconfirmed for the bald area to which the CNP gel had been applied.

FIG. 57 A photographic diagram showing the therapeutic effect when a BNPgel was applied to the left frontal region of alopecia areata case B4(case C9) test subject and a CNP ointment was applied to the rightfrontal region. The test subject faced forward and slightly downward inthe photograph. P denotes the hair loss site before application, Tdenotes the same site at a time when 100 μg/g BNP gel had been appliedfor 3 weeks and application had been suspended for 2 weeks for the leftfrontal region and at a time immediately after 100 μg/g CNP ointment hadbeen applied for 3 weeks to the right frontal region, and T2 denotes thesame site when application had subsequently been suspended for 1 year.The alopecia areata multilocularis was completely cured by applying theBNP gel or the CNP ointment for 3 weeks, and there was no recurrence forover 1 year.

MODES FOR CARRYING OUT THE INVENTION

The present invention is an agent for the treatment or prevention ofalopecia, dandruff, white hair, and seborrheic scalp, the agentcontaining a natriuretic peptide (NP) as an active ingredient, and theagent being for the treatment of alopecia areata, androgenetic alopecia,seborrheic alopecia, alopecia pityroides, female pattern alopecia,postpartum alopecia, senile alopecia, cancer chemotherapy drug-inducedalopecia, and alopecia due to radiation exposure.

The alopecia areata referred to in the present specification is notparticularly limited and means alopecia that is generally clinicallydiagnosed as alopecia areata, in particular alopecia that occurssuddenly without a clear cause in a target having a history of, orcoexisting, immune disease or a target having a genetic background ofimmune disease. Alopecia areata in the present specification includesalopecia that is generally clinically diagnosed as alopecia areata, butis not limited thereto, and includes all types of alopecia in a targethaving an overreaction or abnormality in the immune system, the causebeing unclear other than there being an overreaction or immuneabnormality in the immune system.

The alopecia that is generally clinically diagnosed as alopecia areatais alopecia in which a coin-sized circular to patchy bald area with aclear outline suddenly occurs without any subjective symptoms, prodromalsymptoms, etc., gradually increases in area if it does not spontaneouslycure, and becomes intractable. With regard to the alopecia that isgenerally clinically diagnosed as alopecia areata, during its activestage broken hair, dead hair (black spot within hair follicle), anddiseased hair that easily falls out are observed within and outside thelesion, the configuration of these hair root parts being similar to theshape of an exclamation mark being conclusive for the diagnosis.

The immune disease referred to in the present specification means adisease caused by an overreaction or immune abnormality in the immunesystem, and includes an allergic disease and an autoimmune disease. Theoverreaction or immune abnormality in the immune system means anexcessive immunoreaction or an abnormal immunoreaction that will damagenormal cells of the target itself, and specifically includes an allergicdisease and an autoimmune disease. The overreaction or immuneabnormality in the immune system can be recognized as a history of, orcoexisting, immune disease. The target having an overreaction or immuneabnormality in the immune system referred to in the presentspecification has the same meaning as alopecia having a history of, orcoexisting, immune disease.

The target having a genetic background of an immune disease means atarget for whom there is one having an overreaction or immuneabnormality in the immune system among the genetically-related family.Since it is known that alopecia areata occurs with high frequency whenthere is one having an immune disease among the genetically-relatedfamily, alopecia of unknown cause in a target having a geneticbackground of immune disease can be considered to be alopecia areata.

In the present specification, when there is one having a history of, orcoexisting, allergic disease or autoimmune disease at least among thebiological parents, biological brothers and sisters, biologicalgrandparents, biological children, and biological grandchildren, it isdetermined that it is a target having a genetic background of immunedisease. Furthermore, in the present specification, when there is onehaving a history of, or coexisting, alopecia areata at least among thebiological parents, brothers and sisters, and grandparents, it isdetermined that it is a target having a genetic background of alopeciaareata.

Here, examples of the allergic disease include atopic disease, pollenallergy, food allergy, latex allergy, metal allergy, sick housesyndrome, allergic granulomatous angiitis, food-dependentexercise-induced anaphylaxy, and celiac disease.

The atopic disease includes bronchial asthma, atopic dermatitis,allergic rhinitis, and atopic alopecia. Atopic alopecia means a state inwhich alopecia is caused by exacerbation of atopic dermatitis. Thetarget having an atopic predisposition referred to in the presentspecification means a target for whom at last one of themselves,biological parents, biological brothers and sisters, biologicalgrandparents, biological children, and biological grandchildren has ahistory of bronchial asthma, atopic dermatitis, allergic rhinitis, oratopic alopecia.

Examples of allergens causing an allergic reaction include house dust,mite, cedar, Dactylis, ragweed, and cat fur. The cedar, Dactylis, andragweed referred to here mean cedar pollen, Dactylis pollen, and ragweedpollen respectively. A target that shows an allergic reaction toward atleast one allergen among house dust, mite, cedar, Dactylis, ragweed, andcat fur in an allergen test such as any of a scratch test, anintradermal test, a patch test, and a specific IgE antibody in vitroassay is a target having a genetic background of an immune disease.

Examples of the autoimmune disease include an autoimmune thyroid diseasesuch as Hashimoto's disease, Basedow's disease, or primaryhypothyroidism, vitiligo, systemic lupus erythematosus, rheumatoidarthritis, myasthenia gravis, chronic discoid lupus erythematosus,localized scleroderma, pemphigus, pemphigoid, herpes gestationis, linearIgA bullous dermatosis, acquired epidermolysis bullosa, Sutton's nevus,Guillain Barré syndrome, chronic atrophic gastritis, autoimmunehepatitis, primary biliary cirrhosis, primary sclerosing cholangitis,autoimmune pancreatitis, aortitis, Goodpasture's syndrome, rapidlyprogressive glomerulonephritis, megaloblastic anemia, autoimmunehemolytic anemia, autoimmune neutropenia, idiopathic thrombocytopenicpurpura, idiopathic Addison's disease, insulin-dependent diabetesmellitus, systemic scleroderma, alopecia areata, Vogt-Koyanagi-Haradadisease, autoimmune optic neuropathy, idiopathic azoospermia, habitualabortion, anti-phospholipid antibody syndrome, polymyositis,dermatomyositis, systemic dermatosclerosis, Sjogren syndrome,IgG4-related disease, polyarteritis nodosa, microscopic polyangiitis,allergic granulomatous angiitis, Wegener's granulomatosis, mixedconnective tissue disease, ulcerative colitis, amyotrophic lateralsclerosis, Crohn's disease, collagen disease, relapsing polychondritis,sarcoidosis, stiff person syndrome, adult Still's disease, multiplesclerosis, immune thrombocytopenic purpura, eosinophilic pneumonia, andBehcet's disease.

The autoimmune disease includes an autoimmune disease with skinsymptoms. Examples of the autoimmune disease with skin symptoms includechronic discoid lupus erythematosus, localized scleroderma, pemphigus,pemphigoid, herpes gestationis, linear IgA bullous dermatosis, acquiredepidermolysis bullosa, vitiligo, and Sutton's nevus. The autoimmunedisease with skin symptoms tends to be accompanied by alopecia.

The autoimmune disease includes an autoimmune disease that is liable tobe accompanied by alopecia areata. Examples of the autoimmune diseasethat is particularly liable to be accompanied by alopecia areata includean autoimmune thyroid disease, vitiligo, systemic lupus erythematosus,rheumatoid arthritis, and myasthenia gravis (Non-Patent Document 8).

The androgenetic alopecia referred to in the present specification meanssymptoms in an adult male or female in which hair on the frontal regionand/or crown gradually becomes vellus hair, thinner, and shorter over afew years or longer. As also described in ‘Androgenetic alopeciaDiagnosis and Treatment Guidelines 2010 edition’ (Non-Patent Document2), regardless of the name ‘androgenetic alopecia’, it occurs in femalesas a pattern in which head hair over a relatively wide area of the crownbecomes thin. Since androgenetic alopecia occurs as a result of hairpapilla cells of the scalp of the frontal region or crown reacting with5α dihydrotestosterone and the hair follicles miniaturizing, if in atarget having a genetic background of androgenetic alopecia, hair of thefrontal region and/or crown gradually becomes vellus hair, thin, andshort over a few years or longer, it is androgenetic alopecia.

Having a genetic background of androgenetic alopecia means having agenetic background of male hormone acting on male hormone-sensitive hairfollicles to form vellus hair, and specifically means that it can begenetically diagnosed that hair papilla cells in the scalp of thefrontal region or crown react with 5α dihydrotestosterone to thus causehair follicle miniaturization or means that there is a related male in astate in which head hair of the frontal region or crown has fallen outand the scalp can be seen from 20 years old onward at least amongbiological parents, biological brothers and sisters, biologicalgrandparents, biological children, and biological grandchildren.

The alopecia pityroides referred to in the present specification meansalopecia pityroides in terms of the normal medical meaning, and alopeciadue to dandruff blocking pores to thus cause inflammation is included inalopecia pityroides.

The female pattern alopecia referred to in the present specificationmeans female pattern alopecia in terms of the normal medical meaning,and alopecia caused by the amount of the female hormone estrogendecreasing relative to the amount of androgen in the blood stream isincluded in female pattern alopecia.

The postpartum alopecia referred to in the present specification meanspostpartum alopecia in terms of the normal medical meaning, and alopeciain which hair whose growth phase has been maintained by estrogen entersthe resting phase all at once after childbirth and hair loss increasesis included in postpartum alopecia.

The target referred to in the present specification means any biologicalindividual, preferably a vertebrate, more preferably a mammal, yet morepreferably a rodent such as a mouse, a rat, a Mongolian gerbil, or aguinea pig, a cat family animal such as a cat, a puma, or a tiger, acervid animal such as a deer or an elk, a rabbit, a dog, a mink, asheep, a goat, a cow, a horse, a monkey, or a human, and most preferablya human.

The natriuretic peptide (NP) referred to in the present invention meansan A-type natriuretic peptide (ANP), a B-type natriuretic peptide (BNP),a C-type natriuretic peptide (CNP), a derivative of these NPs, achimeric peptide of 2 or more NPs selected from these NPs, or aderivative of a chimeric peptide of the NPs, and is not particularlylimited as long as it has ANP activity, BNP activity, or CNP activity.It is particularly preferably ANP, BNP, or CNP.

The ANP referred to here means ANP-28 (α-ANP) formed from amino acids,α-ANP[4-28] formed from the 4^(th) to the 28^(th) amino acids of ANP-28,α-ANP[5-28] formed from the 5^(th) to the 28^(th) amino acids of ANP-28,or a derivative thereof, and is not particularly limited as long as ithas ANP activity. The ANP derivatives include, as long as they have ANPactivity, one in which the C-terminal of ANP has been amidated, one inwhich the C-terminal of ANP has been methoxylated, one in whichpolyethylene glycol has been added to ANP, one in which a sugar chainhas been added to ANP, one in which an alkyl chain has been added toANP, and one in which a fatty acid has been added to ANP. The ANP may beβ-ANP, which has a structure that is an antiparallel dimer of ANP-28, ora polymer type γ-ANP having a molecular weight of 13000 formed bycleaving a signal peptide from an ANP precursor. α-ANP and a derivativethereof are particularly preferable. The amino acid sequence of humanANP-28 is shown as the sequence with SEQ ID No: 1 in the presentspecification.

The ANP derivative referred to here means one having preferably 1 to 5,more preferably 1 to 3, and yet more preferably 1 to 2 amino acids inthe ANP-28 amino acid sequence deleted, substituted, or added and havingANP activity, or one having a sequence that is at least 85%, preferablyat least 90%, and yet more preferably at least 95% homologous to theANP-28 amino acid sequence and having ANP activity. The ANP derivativeincludes, as long as it has ANP activity, one in which the C-terminal ofANP has been amidated, one in which the C-terminal of ANP has beenmethoxylated, one in which polyethylene glycol has been added to ANP,one in which a sugar chain has been added to ANP, one in which an alkylchain has been added to ANP, and one in which a fatty acid has beenadded to ANP.

In the present invention, provided that it has ANP activity, any knownANP can be used. Examples thereof include 819 ANP derivates disclosed asAP1 to AP819 in U.S. Pat. No. 5,047,397 or U.S. Pat. Nos. 4,804,650 and32 ANP derivatives disclosed as Compound Nos. 1 to 32 in Tables 1 to 3of U.S. Pat. No. 5,204,328.

Furthermore, the presence or absence of ANP activity may be confirmedeasily by known means and, for example, it may be confirmed by testingcGMP production activity in NPR-A receptor-expressing cells.

As an active ingredient of the present invention, any ANP or derivativethereof can be used, but from the viewpoint of efficacy and availabilityANP-28 is preferable.

The ANP of the present invention can be prepared by chemical synthesisor genetic engineering using a human ANP gene (e.g. Nature. 1984 Vol.312 (5990): 152-155), but since ANP is already commercially available,it may be obtained from the market. Furthermore, it may be obtained forexample as ANP (Human, 1-28) from the Peptide Institute, Inc.

As hereinbefore described, the ANP that can be used in the presentinvention includes purified natural ANP, recombinant ANP produced by aknown genetic engineering method, and ANP produced by a known chemicalsynthesis method (e.g. a solid-phase synthesis method using a peptidesynthesizer). Basic methods such as a gene-recombination technique, asite-specific mutagenesis method, or a PCR technique are publicly knownor well known and are described in, for example, Current Protocols InMolecular Biology; John Wiley & Sons (1998).

The BNP referred to here means BNP-26, BNP-32, or BNP-45, which have 26,32, and 45 amino acids respectively, or a derivative thereof, and is notparticularly limited as long as it has BNP activity. The BNP may be apolymer type γ-BNP formed by cleaving a signal peptide from a BNPprecursor and having a molecular weight of about 13000. It isparticularly preferably BNP-32 or a derivative thereof. The amino acidsequence of human BNP-32 is shown as the sequence with SEQ ID No: 41 inthe present specification. Furthermore, the amino acid sequence of humanBNP-26 is shown as the sequence with SEQ ID No: 82 in the presentspecification.

The BNP derivative referred to here means one having preferably 1 to 5,more preferably 1 to 3, and yet more preferably 1 or 2 amino acids inthe amino acid sequence of BNP-26, BNP-32, or BNP-45 deleted,substituted, or added and having BNP activity, or one having a sequencethat is at least 85%, preferably at least 90%, and more preferably atleast 95% homologous to the BNP-26, BNP-32, or BNP-45 amino acidsequence and having BNP activity. The BNP derivative includes, as longas it has BNP activity, one in which the C-terminal of BNP has beenamidated, one in which the C-terminal of BNP has been methoxylated, onein which polyethylene glycol has been added to BNP, one in which a sugarchain has been added to BNP, one in which an alkyl chain has been addedto BNP, and one in which a fatty acid has been added to BNP.

In the present invention, provided that it has BNP activity, any knownBNP can be used. Examples thereof include a BNP derivative disclosed inpublished Japanese translation 2007-525213 of a PCT application, 29 BNPderivatives disclosed as SEQ ID No: 1 to 29 in U.S. Pat. No. 6,028,055,a BNP derivative disclosed by the Formula of Claim 1 in U.S. Pat. No.5,114,923, BD-NP disclosed in U.S. Pat. No. 6,818,619, and a diureticpolypeptide or a natriuretic polypeptide disclosed in published Japanesetranslation 2010-500032 of a PCT application.

Furthermore, the presence or absence of BNP activity may be confirmedeasily by known means and, for example, it may be confirmed by testingcGMP production activity in NPR-A receptor-expressing cells.

As an active ingredient of the present invention, any of BNP-26, BNP-32,BNP-45, and derivatives thereof can be used, but from the viewpoint ofefficacy and availability BNP-32 is preferable.

The BNP of the present invention can be prepared by chemical synthesisor genetic engineering using a human BNP gene (e.g. JP, A, 5-207891,published Japanese translation 2007-525957 of a PCT application,published Japanese translation 2007-525213 of a PCT application), butsince BNP is already commercially available, it may be obtained from themarket. Furthermore, it may be obtained for example as BNP-32 (human)from the Peptide Institute, Inc.

As hereinbefore described, the BNP that can be used in the presentinvention includes purified natural BNP, recombinant BNP produced by aknown genetic engineering method, and BNP produced by a known chemicalsynthesis method (e.g. a solid-phase synthesis method using a peptidesynthesizer). Basic methods such as a gene-recombination technique, asite-specific mutagenesis method, or a PCR technique are publicly knownor well known and are described in, for example, Current Protocols InMolecular Biology; John Wiley & Sons (1998), JP, A, 5-207891, etc.

The CNP referred to here means CNP-22 having 22 amino acids, CNP-53having 53 amino acids formed by adding 31 amino acid residues to theN-terminal of CNP-22, or a derivative thereof, and is not particularlylimited as long as it has CNP activity. CNP-22 and a derivative thereofare particularly preferable.

The CNP derivative referred to here means one having preferably 1 to 5,more preferably 1 to 3, and yet more preferably 1 or 2 amino acids inthe CNP-22 or CNP-53 amino acid sequence deleted, substituted, or addedand having CNP activity, or one having a sequence that is at least 85%,preferably at least 90%, and yet more preferably at least 95% homologousto the CNP-22 or CNP-53 amino acid sequence and having CNP activity. TheCNP derivative includes, as long as it has CNP activity, one in whichthe C-terminal of CNP has been amidated, one in which the C-terminal ofCNP has been methoxylated, one in which polyethylene glycol has beenadded to CNP, one in which a sugar chain has been added to CNP, one inwhich an alkyl chain has been added to CNP, and one in which a fattyacid has been added to CNP. The amino acid sequence of human CNP-22 isshown as the sequence with SEQ ID No: 81 in the present specification.Furthermore, the amino acid sequence of human CNP-53 is shown as thesequence with SEQ ID No: 83 in the present specification.

In the present invention, provided that it has CNP activity, any knownCNP can be used. Examples thereof include a CNP derivative disclosed inJP, A, 6-9688, CNP derivatives disclosed as VNP (SEQ ID No: 4) and SEQID No: 9 peptides in U.S. Pat. No. 558,310, and CD-NP disclosed in U.S.Pat. No. 6,818,619. Furthermore, the presence or absence of CNP activitymay be confirmed easily by known means and, for example, it may beconfirmed by testing the growth-inhibitory action for vascular smoothmuscle cells or cGMP production activity in NPR-B receptor-expressingcells.

As an active ingredient of the present invention, any of CNP-22, CNP-53,and derivatives thereof can be used, but from the viewpoint ofabsorbability CNP-22, which has a low molecular weight, is preferable.CNP-22 may be prepared by chemical synthesis or genetic engineeringusing a human CNP gene, but may be obtained for example from the PeptideInstitute, Inc. as CNP-22 (human).

The CNP that can be used in the present invention includes purifiednatural CNP, recombinant CNP produced by a known genetic engineeringmethod, and CNP produced by a known chemical synthesis method (e.g. asolid-phase synthesis method using a peptide synthesizer). Basic methodssuch as a gene-recombination technique, a site-specific mutagenesismethod, or a PCR technique are publicly known or well known and aredescribed in, for example, Current Protocols In Molecular Biology; JohnWiley & Sons (1998), JP, A, 5-207891, etc.

The origin of a natriuretic peptide is not particularly limited as longas it has CNP activity, BNP activity, or ANP activity, but it ispreferably mammal (including human)- or bird-derived NP, more preferablyhuman-, monkey-, mouse-, rat-, or pig-derived NP, and particularlypreferably human-derived NP.

With regard to amino acids that can be substituted in the ANPderivatives, BNP derivatives, and CNP derivatives, it is desirable tocarry out conservative amino acid substitution. Conservative amino acidsare classified according to polarity or type of electric charge. Forexample, nonpolar uncharged type amino acids include glycine, alanine,valine, leucine, isoleucine, proline, etc., aromatic amino acids includephenylalanine, tyrosine, and tryptophan, polar uncharged type aminoacids include serine, threonine, cysteine, methionine, asparagine,glutamine, etc., negatively charged amino acids include aspartic acidand glutamic acid, and positively charged amino acids include lysine,arginine, and histidine. As described above, amino acid substitution isdesirably carried out such that conservative amino acids belonging tothe same class are substituted for each other. However, when anothernonpolar uncharged type amino acid is substituted for proline or whenproline is substituted for a nonpolar uncharged type amino acid otherthan proline, it is necessary to note that proline does not have asterically flexible structure. Furthermore, when another polar unchargedtype amino acid is substituted for cysteine or when cysteine issubstituted for a polar uncharged type amino acid other than cysteine,it is necessary to note that cysteine can form a disulfide bond withanother cysteine.

Moreover, when a natriuretic peptide (NP) is referred to in the presentspecification, a chimeric peptide of two or more natriuretic peptides(NPs) selected from ANP, BNP, and CNP is also included. That is, when anNP is referred to in the present specification, it also means

a chimeric peptide of 2 or more natriuretic peptides (NPs) selected fromANP, BNP, and CNP, the chimeric peptide forming a cyclic structure viaan intramolecular disulfide bond,

the CNP being a peptide selected from the group consisting of CNP-22,CNP-53, a peptide containing any amino acid sequence having 5 or morecontiguous amino acids from the CNP-22 amino acid sequence in which any1 to 5 amino acids have been deleted, substituted, or added, and apeptide containing any amino acid sequence having 5 or more contiguousamino acids from the CNP-53 amino acid sequence in which any 1 to 5amino acids have been deleted, substituted, or added,

the BNP being a peptide selected from the group consisting of BNP-26,BNP-32, BNP-45, a peptide containing any amino acid sequence having 5 ormore contiguous amino acids from the BNP-26 amino acid sequence in whichany 1 to 5 amino acids have been deleted, substituted, or added, apeptide containing any amino acid sequence having 5 or more contiguousamino acids from the BNP-32 amino acid sequence in which any 1 to 5amino acids have been deleted, substituted, or added, and a peptidecontaining any amino acid sequence having 5 or more contiguous aminoacids from the BNP-45 amino acid sequence in which any 1 to 5 aminoacids have been deleted, substituted, or added,

the ANP being ANP-28 or a peptide containing any amino acid sequencehaving 5 or more contiguous amino acids from the ANP amino acid sequencein which any 1 to 5 amino acids have been deleted, substituted, oradded, and

the chimeric peptide having CNP activity, BNP activity, or ANP activity,or a derivative thereof.

A preferred chimeric peptide of the natriuretic peptides (NPs) in thepresent invention is a chimeric peptide of CNP and BNP. That is, it ispreferable to use

a chimeric peptide of CNP and BNP, the chimeric peptide forming a cyclicstructure via an intramolecular disulfide bond,

the CNP being a peptide selected from the group consisting of CNP-22,CNP-53, a peptide containing any amino acid sequence having 5 or morecontiguous amino acids from the CNP-22 amino acid sequence in which any1 to 5 amino acids have been deleted, substituted, or added, and apeptide containing any amino acid sequence having 5 or more contiguousamino acids from the CNP-53 amino acid sequence in which any 1 to 5amino acids have been deleted, substituted, or added,

the BNP being a peptide selected from the group consisting of BNP-26,BNP-32, BNP-45, a peptide containing any amino acid sequence having 5 ormore contiguous amino acids from the BNP-26 amino acid sequence in whichany 1 to 5 amino acids have been deleted, substituted, or added, apeptide containing any amino acid sequence having 5 or more contiguousamino acids from the BNP-32 amino acid sequence in which any 1 to 5amino acids have been deleted, substituted, or added, and a peptidecontaining any amino acid sequence having 5 or more contiguous aminoacids from the BNP-45 amino acid sequence in which any 1 to 5 aminoacids have been deleted, substituted, or added, and

the chimeric peptide having CNP activity or BNP activity, or aderivative thereof.

When CNP or BNP is referred to in the present specification, this meanseither one of CNP or BNP and also means a chimeric peptide of CNP andBNP.

The origin of CNP-22 and CNP-53 is not particularly limited as long asit has CNP activity, but it is preferably mammal (including human)- orbird-derived CNP, more preferably human-, monkey-, mouse-, rat-, orpig-derived CNP, and particularly preferably human-derived CNP.Similarly, the origin of BNP-26, BNP-32, and BNP-45 is not particularlylimited as long as it has BNP activity, but it is preferably mammal(including human)- or bird-derived BNP, more preferably human-, monkey-,mouse-, rat-, or pig-derived BNP, and particularly preferablyhuman-derived BNP. Similarly, the origin of ANP-28 is not particularlylimited as long as it has ANP activity, but it is preferably mammal(including human)- or bird-derived ANP, more preferably human-, monkey-,mouse-, rat-, or pig-derived ANP, and particularly preferablyhuman-derived ANP.

Furthermore, the derivative of a chimeric peptide of 2 or more NPsselected from ANP, BNP, and CNP referred to here means one havingpreferably 1 to 5, more preferably 1 to 3, and yet more preferably 1 or2, amino acids in the NP chimeric peptide amino acid sequence deleted,substituted or added and having CNP activity or BNP activity.

The amino acids that can be substituted in a derivative of a chimericpeptide of natriuretic peptide (NP) derivatives are the same as theamino acids that can be substituted with respect to the ANP derivative,BNP derivative, and CNP derivative.

Furthermore, the derivative of the chimeric peptide of the natriureticpeptide (NP) derivatives includes, as long as it has CNP activity, BNPactivity, or ANP activity, one in which the C-terminal of the chimericpeptide of the NPs has been amidated, one in which the C-terminal of thechimeric peptide of the NPs has been methoxylated, one in whichpolyethylene glycol has been added to the chimeric peptide of the NPs,one in which a sugar chain has been added to the chimeric peptide of theNPs, one in which an alkyl chain has been added to the chimeric peptideof the NPs, and one in which a fatty acid has been added to the chimericpeptide of the NPs.

Furthermore, the amino acid sequence of human ANP peptide represented bySEQ ID No: 1, the amino acid sequence of human BNP peptide representedby SEQ ID No: 41, and the amino acid sequence of human CNP peptiderepresented by SEQ ID No: 81 have, as shown in FIG. 1, four mutuallydifferent sequences separated by three common sequences represented by‘CFG’, ‘DRI’, and ‘SGLGC’ amino acid sequences respectively. Therefore,as chimeric peptides of 2 or more NPs selected from ANP, BNP, and CNP,in accordance with combinations of these four mutually differentsequences, at least 78 types of chimeric peptides represented by SEQ IDNo: 2 to 40 and SEQ ID No: 42 to 80 can be cited as examples. It can beexpected that these chimeric peptides and derivatives thereof will haveproperties in common with ANP, BNP, and CNP. That is, these chimericpeptides and derivatives thereof may be used as an active ingredient ofthe agent for the treatment of alopecia of the present invention.

As described above, in the present invention, a chimeric peptide of anyknown natriuretic peptides (NPs) or a derivative thereof can be usedprovided that it has ANP activity, BNP activity, or CNP activity. Forexample, it may be an aquaretic polypeptide or a natriuretic polypeptidedisclosed as ABC-NP, ABC-NP1, BC-NP, etc. in published Japanesetranslation 2010-502231 of a PCT application, or a chimeric peptidedisclosed as BD-NP (SEQ ID No: 1) or CD-NP (SEQ ID No: 2) in U.S. Pat.No. 6,818,619.

The presence or absence of ANP activity, BNP activity, or CNP activitycan be easily confirmed by known means, and it can be confirmed by, forexample, testing cGMP production activity in NPR-A receptor-expressingcells or NPR-B expressing cells.

All of ANP, BNP, CNP, derivatives of these NPs, and chimeric peptides of2 or more NPs selected from these NPs are known and may be prepared bychemical synthesis or genetic engineering.

The disease to which the agent for the treatment of alopecia of thepresent invention is applied is not particularly limited as long as itis so-called alopecia in which hair thins or falls out. The agent forthe treatment of alopecia of the present invention may be applied tovarious types of alopecia.

Examples of alopecia to which the agent for the treatment of alopecia ofthe present invention can be applied include alopecia areata,androgenetic alopecia, postpartum alopecia, female pattern alopecia,seborrheic alopecia, alopecia pityroides, and senile alopecia; from theviewpoint of the curing effect, alopecia areata, androgenetic alopecia,postpartum alopecia, and female pattern alopecia are preferable, andalopecia areata is particularly preferable.

Androgenetic alopecia may be either androgenetic alopecia in a male orandrogenetic alopecia in a female. In accordance with use of the agentfor the treatment of alopecia of the present invention, it is possibleto regenerate effectively head hair on the frontal region or the crown.

With regard to alopecia areata, it may be any of standard alopeciaareata monolocularis, standard alopecia areata multilocularis, alopeciatotalis, alopecia universalis, and alopecia ophiasis, and the severitythereof may be any of S1 to S5 and B0 to B2.

The agent for the treatment of alopecia of the present inventionexhibits excellent effectiveness and safety as a drug for the treatmentof intractable alopecia ophiasis, which is considered to be difficult tocure or put into remission for a long time, and is effective.

The meaning of the terminology and characteristics of the symptoms ofthese various types of alopecia are as described in the Background Artsection above.

The agent for the treatment of alopecia of the present inventioncontains as an active ingredient an A-type natriuretic peptide (ANP), aB-type natriuretic peptide (BNP), a C-type natriuretic peptide (CNP), aderivative of these natriuretic peptides (NPs), a chimeric peptide of 2or more NPs selected from these NPs, or a derivative of a chimericpeptide of 2 or more NPs selected from these NPs, and its route ofadministration and dosage form are not particularly limited.

The agent for the treatment of alopecia of the present invention may, inaddition to ANP, BNP, CNP, a derivative of these NPs, a chimeric peptideof 2 or more NPs selected from these NPs, or a derivative thereof,further contain as a combination a steroid drug, an antihistamine drug,a vasodilator, a male hormone activity inhibitor, a female hormone drug,an antibiotic, an antifungal agent, pentadecane, cytopurine(6-benzylaminopurine), t-flavanone, adenosine, cepharanthin, Glycyron(registered trademark), which is a complex of glycyrrhizin, methionine,and glycine, cyclosporin A, Keishikaryukotsuboreito, Hangekobokuto,biotin, Anthralin, or a tricyclic antidepressant, or may be used at thesame time as these.

As the steroid drug, diflorasone, betamethasone, dexamethasone,clobetasol, prednisolone, mometasone, methylprednisolone, Deprodone,difluprednate, fluocinonide, amcinonide, triamcinolone, difluprednate,or hydrocortisone is preferable, and betamethasone or clobetasol isparticularly preferable.

As the antihistamine drug, azelastine, oxatomide, fexofenadine,emedastine, ebastine, cetirizine, bepotastine, olopatadine, orloratadine is preferable.

As the vasodilator, minoxidil or carpronium chloride is preferable. Asthe male hormone activity inhibitor, finasteride is preferable.

As the female hormone drug, estrogen, estradiol, or progesterone ispreferable.

As the antibiotic, gentamicin is preferable. As the antifungal agent,amphotericin B, nystatin, ketoconazole, terbinafine, flucytosine,fluconazole, itoraconazole, griseofulvin, or micafungin is preferable.

The agent for the treatment of alopecia of the present invention can bemade into a preparation by generally used techniques. Examples of thepreparation configuration include an external preparation.

The dosage form of the agent for the treatment of alopecia of thepresent invention is preferably an external preparation (percutaneousabsorbent) such as a gel, an ointment, or a liquid.

The external preparation is not particularly limited and the presentagent may be directly applied, sprayed, or put as a patch onto arequired site (affected part) of the skin. The configuration of theagent for the treatment of alopecia of the present invention ispreferably an external preparation such as an ointment, gel, cream,lotion, liquid, spray, gel spray, foam, patch, shampoo, treatment, scalptreatment, or tonic, it is particularly preferably an ointment, gel,cream, or liquid from the viewpoint of simplicity of application, and itis yet more preferably a gel, ointment, or liquid.

These external preparations may be easily obtained in accordance withpublicly or well known methods by formulating a natriuretic peptide (NP)as an active ingredient or principal agent, a pharmaceuticallyacceptable base, and various types of additives as necessary.

The gel (suspension base) may be any of a hydrogel, an anhydrous gel, ora gel formed from a swellable gel-forming material and having a lowwater content. Furthermore, either a hydrogel base or a lyogel base maybe used, but a transparent hydrogel having an inorganic or organicmacromolecular base is preferable. In the same way as for a preparationcontaining an oil or a fat, the gel itself is not absorbed by the skin.

A hydrogel base is free from fat, has a consistency similar to that ofan ointment, and aims to increase the percutaneous absorbability of themedicinal agent, and a lyogel base is formed by suspending stearylalcohol, etc. in propylene glycol to form a gel and has excellentpercutaneous absorbability and hygroscopicity. The gel may be used as agel spray by charging a spray container therewith.

The gel of the present invention may be a gel in which a natriureticpeptide (NP) as an active ingredient is uniformly dispersed in ahydrophilic gel base containing a carboxyvinyl polymer, sodiumpolyacrylate, sodium polyacrylate, a (vinyl methyl ether/ethyl maleate)copolymer, polymethacrylate, propylene glycol, etc. Examples of such agel include a gel formed by uniformly dispersing in a commerciallyavailable long-lasting water retention agent such as Lubrajel NP,Lubrajel CG, Lubrajel DV, Lubrajel MS, Lubrajel OIL, Lubrajel TW, orLubrajel DS, which are commercial products from ISP Japan Ltd.

One specific example of the ‘gel’ of the present invention is a gelpreparation prepared in accordance with Example 2.

The liquid of the present invention is one in which a natriureticpeptide (NP) as an active ingredient is dissolved in alcohol, propyleneglycol, polyethylene glycol, water, etc. as a base, and is preferably aliquid formed from an aqueous solution in which a natriuretic peptide isdissolved in physiological saline. The aqueous solution may contain asmall amount of organic base such as alcohol, propylene glycol, orpolyethylene glycol in addition to physiological saline.

In this case, in order to ensure the biological utilization rate andprovide a more effective liquid preparation, a natriuretic peptide isused as an active ingredient, an acidic solution may be formed bycombining it with one type or two or more types from the groupconsisting of butyric acid, lactic acid, phosphoric acid, glycine,citric acid, hydrochloric acid, propionic acid, butyric acid, benzoicacid, and salts thereof, or a polar organic solution may be formed bycombining it with one type or two or more types from the groupconsisting of an alcohol and/or N-methyl-2-pyrrolidine,dimethylformamide, dimethylsulfoxide, and methylparaben, and the pH maybe adjusted to 3.0-7.0.

The ointment preparation of the present invention may be either anoleaginous base or a water-soluble base, and both may easily be obtainedin accordance with a known method. An oleaginous base such as Vaselinecauses little irritation, has no odor, and is excellent in terms of skinprotecting action, softening action, crust removing action, granulation,and epithelization promoting action. The water-soluble base is anointment mainly containing a Macrogol base and has a strong effect inabsorbing and removing aqueous secretions.

One specific example of the ‘ointment’ of the present invention is anointment prepared in accordance with Example 2.

The cream (emulsion base) may be an oil-in-water base (O/W) (vanishingcream) or a water-in-oil base (W/O) (cold cream). The oil-in-water basehas a smaller amount of oil-soluble component than water-solublecomponent, and has the advantage that when applied the whiteness of thecream seems to disappear. It also spreads well, has a good feeling whenused, even on sweaty skin, and has excellent cosmetic aspects.Furthermore, since it has excellent absorbability toward the skin,application to a chronic hypertrophic lesion is good. The water-in-oilbase contains a smaller amount of water-soluble component thanoil-soluble component and is also called a cold cream since when appliedto and spread on the skin it exhibits a cooling action.

The lotion means a liquid external preparation in which a natriureticpeptide is dissolved or uniformly dispersed in a liquid. Since anointment or a cream sticks to the hair, a lotion is suitable for use onhead hair, etc. The configuration of the lotion may be any of asuspension lotion base, an emulsion lotion, and a solution lotion base.

The patch promotes absorption of a medicinal agent by making a componentcontaining a natriuretic peptide adhere to the patch and utilizing theairtightness of the patch. Applying a patch enables scratching to beprevented.

The spray is used by making a solution of a natriuretic peptide andspraying by means of gas pressure or a hand pushing operation. It isconvenient when used over a wide area.

The foam is a spray that is released in the form of a foam by making asolution of a natriuretic peptide and adding a surfactant. The foam isexcellent in terms of adhesion to the scalp.

The shampoo is a dosage form that has a natriuretic peptide mixed ordissolved in a shampoo and that can apply the medicinal agent at thesame time as washing the hair. Since a shampoo is a surfactant, it hasan effect in making a natriuretic peptide penetrate into the scalp. Inseborrheic alopecia in particular, a shampoo is advantageous since itcan also remove excessive sebum.

The treatment is a dosage form that has a natriuretic peptide mixed ordissolved in a treatment used when washing hair and that can apply themedicinal agent at the same time as treatment being carried out. Thetreatment can supplement moisture and oil content in the scalp at thesame time as application of the medicinal agent. In alopecia pityroidesin particular, a treatment is advantageous since it can also moisturizeand supplement the oil content in the scalp at the same time asapplication of the medicinal agent.

The scalp treatment is a treatment agent in which a component designedto moisturize or supplement the oil content in the scalp is particularlyformulated. A scalp treatment often contains various plant extractcomponents such as rosemary extract, soapberry extract, or coconutextract.

The tonic may be prepared by adding a natriuretic peptide to 50% to 70%alcohol and water as a substrate, a component having an action ofkeeping head hair and the scalp healthy such as hinokitiol, panthenol,or Swertia Japonica extract, and dipotassium glycyrrhizinate, whichexhibits a female hormone-like action, together with a microbicidalagent, a moisturizing component such as glycerol, salicylic acid, whichmakes it easy to remove dandruff, menthol, which prevents itchiness andgives a refreshing feel, a fragrance, etc. A tonic is advantageous as adosage form of an agent for the treatment of alopecia that is oftenaccompanied by itchiness since it is a dosage form that is used afterwashing hair in order to prevent dandruff and head odor, maintain thecleanliness of head hair, prevent itchiness and perspiration damage, andremove unpleasant symptoms related to head hair.

As described above, the agent for the treatment of alopecia of thepresent invention is a percutaneous external preparation formed byformulating appropriate amounts of a natriuretic peptide, various typesof base, and additives as necessary. In order to exhibit efficacy as anexternal preparation, the way in which the natriuretic peptide as anactive ingredient applied to the skin surface reaches a lesion area atan effective concentration and is maintained is important. Therefore,the dosage form and the base may be selected appropriately according tothe symptoms or the patient.

Furthermore, an additive may be used as appropriate according to theintended application. As the additive, those below can be used.

Vaseline: may be used as a base of an ointment preparation. Itsviscosity and consistency changes depending on temperature and thehardness varies between winter and summer, but it is one of the safestbases. There are yellow Vaseline and white Vaseline, which has a highdegree of purification, and either may be used.

Propylene glycol: may be used as a solvent for a drug, a solutionadjuvant, or a base.

Paraffin: may be used when the consistency of an ointment is adjusted.Since it is comparatively easy to emulsify, it may be used as anoleaginous agent in production of a cream.

Beeswax (white beeswax): processed beehive wax, and may be used as aJapanese Pharmacopoeia simple ointment by formulating with a plant oil.White beeswax is formed by bleaching beeswax so as to improve color andodor.

Macrogol: a mixture of polyethylene glycols having different molecularweights. It has excellent solubility and miscibility for a medicinalagent, absorbs water well, and is therefore suitable when absorbing andremoving a liquid leached out from the mucous membrane or the affectedlesion area.

Stearyl alcohol: may be used in an emulsion lotion.

Isopropanol: may be used as a solvent, a solution adjuvant, etc.

Benzyl alcohol: may be used as a solution adjuvant, a preservative, etc.

Parahydroxybenzoic acid esters (parabens): may be used as an antiseptic,a preservative, or a stabilizer.

Gelled hydrocarbon: generally called a ‘Plastibase’ and formed bygelling (semi-solidifying) liquid paraffin using polyethylene.

Citric acid and sodium citrate: may be used as a buffer or a pHadjusting agent.

Squalene: used as a base and has a slightly less oily feel and lessstickiness than liquid paraffin. It can be used widely in emulsionlotions as well as in creams.

Lanolins: fats obtained from sheep wool, useful for improvement of skinsuppleness although they have a problem with color and odor.

Glycerol: may be formulated into a cream, etc. as a moisturizing agent.

Polyoxyethylene hardened castor oil: may be used as an emulsifyingagent, a solubilizing agent, etc.

Sorbitan fatty acid ester and glycerol fatty acid ester: may be used asan emulsifying agent, etc.

The agent for the treatment of alopecia of the present invention mayfurther contain a moisturizing agent (skin softening agent), asymptom-relieving agent, etc., which are described below.

Moisturizing agent (skin softening agent): a moisturizing agent providesmoisture and oil to the skin. A moisturizing agent is most effective ifit is used when the skin is already wet, as is the case immediatelyafter a bath or shower. Components contained in the moisturizing agentare glycerol, mineral oil, Vaseline, etc. With regard to the form andtype of the moisturizing agent, there is a lotion, a cream, an ointment,a bath oil, etc. One containing urea, lactic acid, or glycolic acid isexcellent in terms of moisturizing effect.

Symptom-relieving agent: many skin diseases are accompanied byitchiness. Itchiness and a slight degree of pain can be relieved byformulating a sedative, specific examples thereof including chamomile,eucalyptus, camphor, menthol, zinc oxide, talc, glycerol, and Calamine.In order to suppress itchiness due to allergy, an antihistamine agentsuch as diphenhydramine may be added.

As hereinbefore described, when producing the agent for the treatment ofalopecia of the present invention, various types of base, moisturizingagent, UV absorbing agent, alcohol, chelate, pH adjusting agent,antiseptic, thickener, colorant, fragrance, filler, excipient,disintegrant, extending agent, binder, film-forming agent, solubilizingagent, suspending agent, buffer, stabilizer, preservative, surfactant,antioxidant, dispersant, emulsifying agent, solvent, solution adjuvant,etc. may be formulated in any combination. Furthermore, in addition to anatriuretic peptide, which is the main medicinal component, varioustypes of medicinal agents such as an anti-inflammatory and analgesicagent, a microbicidal agent, a vitamin, and a skin softening agent maybe appropriately formulated as necessary.

When the skin external preparation composition of the present inventionis used as a skin texture improvement agent, it may be used as a skincare cosmetic or a quasi-drug, and specific examples of the applicationform include a cream, a foam, a cosmetic lotion, a pack, a skinsoftener, an emulsion, a foundation, a makeup base, an essence, a soap,a liquid washing agent, a bath agent, a sunscreen cream, a sun oil, anda spray type liquid. All thereof may be produced easily by applying apublicly or well known preparation technique.

As representative preparation examples of the present agent for thetreatment of alopecia, production of a gel, an ointment, and a liquid isnow explained.

A gel may be obtained, in accordance with a publicly or well knownmethod, by dissolving an appropriate amount of natriuretic peptide indistilled water or physiological saline to give an aqueous solution, andmixing and stirring this with a publicly or well known gel base. Whenthe concentration of the natriuretic peptide is no greater than 1 μg/g,the effect is not sufficient, and a sufficient effect can be obtainedwithout formulating it over 1000 μg/g. Preparation is carried out suchthat the final concentration of the natriuretic peptide in the gel ispreferably 1 to 1000 μg/g, more preferably 10 to 500 μg/g, yet morepreferably 20 to 300 μg/g, even more preferably 30 to 200 μg/g, andparticularly preferably 50 to 100 μg/g.

Examples of a gel base formed from a macromolecular inorganic componentinclude hydrated or water-absorbing silicates such as aluminum silicate,for example bentonite, magnesium aluminum silicate, and colloidalsilica. As a gel base formed from a macromolecular organic material, anatural, semi-synthetic, or synthetic polymer may be used. Examples ofthe natural and semi-synthetic polymers include polysaccharides such ascellulose, starch, tragacanth, gum arabic, xanthan gum, agar-agar,gelatin, alginic acid, a salt thereof such as sodium alginate, and aderivative thereof, a lower alkyl cellulose such as methylcellulose orethylcellulose, and a carboxy- or hydroxy-lower-alkyl cellulose such ascarboxymethylcellulose or hydroxypropylcellulose. Examples of thesynthetic gel base include polyvinyl alcohol, polyvinylpyrrolidone,polyacrylic acid, and polymethacrylic acid. Furthermore, a gel baseformed by carrying out uniform dispersion in a commercially availablelong-lasting water retention agent such as Lubrajel NP, Lubrajel CG,Lubrajel DV, Lubrajel MS, Lubrajel OIL, Lubrajel TW, or Lubrajel DS,which are commercial products from ISP Japan Ltd., may be used. Withregard to these gel bases, one type thereof or a gel base mixture of twoor more types thereof may be used.

The ointment (Vaseline preparation) may be obtained, in accordance witha publicly or well known method, by dissolving an appropriate amount ofnatriuretic peptide in distilled water or physiological saline to givean aqueous solution, and mixing and stirring this with a publicly orwell known Vaseline. When the concentration of the natriuretic peptideis no greater than 1 μg/g, the effect is not sufficient, and asufficient effect can be obtained without formulating it over 1000 μg/g.Preparation is carried out so that the final concentration of thenatriuretic peptide in the ointment is preferably 1 to 1000 μg/g, morepreferably 10 to 500 μg/g, yet more preferably 20 to 300 μg/g, even morepreferably 30 to 200 μg/g, and particularly preferably 50 to 100 μg/g.

The liquid may be prepared by, for example, dissolving as a main agent0.01 mg to 10 mg of human ANP (1-28) (Peptide Institute, Inc.), humanBNP-32 (Peptide Institute, Inc.), or human CNP-22 (Peptide Institute,Inc.) in 10 mL of physiological saline, thus giving a liquid having anatriuretic peptide concentration of 1 to 1000 μg/g. Since the specificgravity of water is 1, in this case the ANP concentration, the BNPconcentration, and the CNP concentration are 1 to 1000 μg/g ratio byweight. When the concentration of the natriuretic peptide is no greaterthan 1 μg/g, the effect is not sufficient, and a sufficient effect canbe obtained without formulating it over 1000 μg/g. The concentration ofthe natriuretic peptide in the aqueous solution preparation ispreferably 1 to 1000 μg/g, more preferably 10 to 500 μg/g, yet morepreferably 20 to 300 μg/g, even more preferably 30 to 200 μg/g, andparticularly preferably 50 to 100 μg/g.

A percutaneous absorption adjuvant may be added as desired. Examples ofthe percutaneous absorption adjuvant include acetic acid, sodiumacetate, limonene, menthol, salicylic acid, hyaluronic acid, oleic acid,N,N-diethyl-m-toluamide, n-butyl stearate, benzyl alcohol, isopropylmyristate, isopropyl palmitate, polypropylene glycol, crotamiton,diethyl sebacate, N-methylpyrrolidone, N-ethylpyrrolidone, and laurylalcohol. Furthermore, an antiseptic, an antioxidant, etc. may be addedas desired.

The concentration of the natriuretic peptide in the present agent forthe treatment of alopecia may be selected as appropriate while takinginto consideration symptoms, age, dosage form, etc. The concentration ofthe natriuretic peptide is preferably 1 to 1000 μg/g relative to theexternal preparation such as a liquid, a gel, an ointment, or a lotion,more preferably 10 to 500 μg/g, yet more preferably 20 to 300 μg/g, evenmore preferably 30 to 200 μg/g, and particularly preferably 50 to 100μg/g. It is preferable to use one with a concentration of 20 to 100 μg/gfor a young patient or a patient with weak skin. Since the solution usedin the liquid of the present invention has a specific gravity ofsubstantially 1, when the concentration of ANP, BNP, or CNP in theliquid is expressed using units of μg/g, it has the same meaning as thatwhen the concentration of the natriuretic peptide is expressed usingunits of μg/mL.

Administration of the present agent for the treatment of alopeciadepends on symptoms, age, agent form, etc., but it is typically twice aday and the period of administration is from 7 days to 28 days. In thecase of alopecia in the chronic stage where symptoms are fixed, it isdesirable to carry out administration for about 28 days, but in theacute stage administration for about 7 days is sufficient. In general,it is desirable to carry out administration for at least 14 days for anadult but administration for about 7 days is sufficient for a minor.

The present invention is explained below by reference to Examples, butthe present invention is in no way limited to these Examples, etc.

EXAMPLES Example 1 Diagnosis and Evaluation of Test Subjects

First, prior to administration of a natriuretic peptide preparation,diagnosis and evaluation of test subjects were carried out. Methods forthe diagnosis and evaluation of test subjects were as follows.

1. Diagnosis of Test Subject;

The test subjects were patients with alopecia areata, androgeneticalopecia, postpartum alopecia, female pattern alopecia, alopeciapityroides, and senile alopecia. Diagnosis and treatment of these testsubjects were carried out by the present inventors as physicians.

2. Evaluation of Symptoms;

Clinical classification of alopecia was carried out in accordance withthe Classification of the Japanese Dermatological Association, andevaluation of the severity of alopecia areata was carried out inaccordance with the above-mentioned ‘USA Alopecia Areata EvaluationGuidelines’, the area of hair loss being classified using 6 grades of S0to S5 and the hair loss site being classified using 3 grades of B0 toB2.

3. Test Method;

In general, in order to confirm the effect of an external medicine in anindividual case, a method involving separate left and right applicationsis desirable. The separate left and right application method is a methodin which, for example, an external medicine containing an activeingredient to be tested is applied to the left-hand side of an affectedsite, and an external medicine not containing the active ingredient isapplied to the right-hand side, thus enabling a therapeutic effect to beconfirmed. With regard to the test of the preparation of the presentinvention, a preparatory test was carried out in accordance with theseparate left and right applications method. However, taking intoconsideration medical ethics, the preparatory test in accordance withthe separate left and right applications method was minimized, and whena preparatory test was not carried out, a therapeutic effect wasevaluated by comparison between that before application and that afterapplication.

Example 2 1. Production of Gel

Preparation of a gel containing an NP as an active ingredient wascarried out by weighing, as a main agent, 3 mg of any one of humanANP(1-28) (Peptide Institute, Inc.), human BNP-32 (Peptide Institute,Inc.), and human CNP-22 (Peptide Institute, Inc.), dissolving this in 3mL of purified water to give an NP solution having a concentration of1000 μg/mL, and mixing 1 mL of this solution with 9 g of Lubrajel NP(ISP Japan Ltd.) by uniform stirring, thus giving an ANP gel, a BNP gel,and a CNP gel having a concentration of 100 μg/g.

Similarly, 500 μL of the NP solution having a concentration of 1000μg/mL obtained above was diluted with 500 μL of physiological saline tothus adjust the concentration to 500 μg/mL, and 1 mL of this solutionwas mixed with 9 g of Lubrajel NP (ISP Japan Ltd.) by uniformlystirring, thus giving an ANP gel, a BNP gel, and a CNP gel having aconcentration of 50 μg/g.

2. Production of Ointment (Vaseline Preparation)

Preparation of an ointment (Vaseline preparation) containing an NP as anactive ingredient was carried out by weighing, as a main agent, 3 mg ofany one of human ANP(1-28) (Peptide Institute, Inc.), human BNP-32(Peptide Institute, Inc.), and human CNP-22 (Peptide Institute, Inc.),dissolving this in 3 mL of physiological saline to give an NP solutionhaving a concentration of 1000 μg/mL, and mixing 1 mL of this solutionwith 9 g of Japanese pharmacopoeia white Vaseline by uniform stirring athigh speed, thus giving an ANP ointment, a BNP ointment, and a CNPointment having a concentration of 100 μg/g.

Similarly, 500 μL of the NP solution having a concentration of 1000μg/mL obtained above was diluted with 500 μL of physiological saline tothus adjust the concentration to 500 μg/mL, and 1 mL of this solutionwas mixed with 9 g of Japanese pharmacopoeia white Vaseline by uniformlystirring at high speed, thus giving an ANP ointment, a BNP ointment, anda CNP ointment having a concentration of 50 μg/g.

Similarly, 300 μL of the BNP solution having a concentration of 1000μg/mL obtained above was diluted with 700 μL of physiological saline tothus adjust the concentration to 300 μg/mL, and 1 mL of this solutionwas mixed with 9 g of Japanese pharmacopoeia white Vaseline by uniformlystirring at high speed, thus giving a BNP ointment having aconcentration of 30 μg/g.

3. Production of BNP:Betamethasone:Gentamicin Combination

Preparation of a BNP, betamethasone, and gentamicin combination wascarried out by mixing Dermosol (trademark) G lotion (Dermosol-G Lotion)manufactured by Iwaki Seiyaku Co., Ltd. with the BNP gel obtained in ‘1.Production of gel’ above at equal volumes. In the present specification,this is called a BNP:betamethasone:gentamicin combination. SinceDermosol G lotion contains betamethasone valerate at a concentration of1200 μg/mL and gentamicin sulfate at a concentration of 1000 μg/mL,mixing the CNP gel having a concentration of 100 μg/g and Dermosol Glotion at a 1:1 ratio by volume gave a combination containing CNP at aconcentration of 50 μg/g, betamethasone valerate at a concentration of600 μg/mL, and gentamicin sulfate at a concentration of 500 μg/mL. Inthe present specification, this is called a 50 μg/g BNP:600 μg/mLbetamethasone:500 μg/mL gentamicin combination.

4. Production of CNP:Betamethasone:Gentamicin Combination

Preparation of a CNP, betamethasone, and gentamicin combination wascarried out by mixing Dermosol (trademark) G lotion (Dermosol-G Lotion)manufactured by Iwaki Seiyaku Co., Ltd. with the CNP gel obtained in ‘1.Production of gel’ above at equal volumes. In the present specification,this is called a CNP:betamethasone:gentamicin combination. SinceDermosol G lotion contains betamethasone valerate at a concentration of1200 μg/mL and gentamicin sulfate at a concentration of 1000 μg/mL,mixing the CNP gel having a concentration of 100 μg/g and Dermosol Glotion at a 1:1 ratio by volume gave a combination containing CNP at aconcentration of 50 μg/g, betamethasone valerate at a concentration of600 μg/mL, and gentamicin sulfate at a concentration of 500 μg/mL. Inthe present specification, this is called a 50 μg/g CNP:600 μg/mLbetamethasone:500 μg/mL gentamicin combination

Similarly, mixing the CNP gel having a concentration of 50 μg/g andDermosol G lotion at a 1:1 ratio by volume gave a combination containingCNP at a concentration of 25 μg/g, betamethasone valerate at aconcentration of 600 μg/mL, and gentamicin sulfate at a concentration of500 μg/mL. In the present specification, this is called a 25 μg/gCNP:600 μg/mL betamethasone:500 μg/mL gentamicin combination.

5. Production of BNP:Clobetasol Combination

A combination of BNP and clobetasol propionate was prepared by mixingDermovate (trademark) Scalp Lotion 0.05% (Dermovate Scalp Lotion 0.05%)manufactured by GlaxoSmithKline plc and the BNP gel obtained in ‘1.Production of gel’ above at equal volumes. In the present specification,this is called a BNP:clobetasol combination. Since Dermovate ScalpLotion 0.05% contains clobetasol propionate at a concentration of 500μg/g, mixing the BNP gel having a concentration of 100 μg/mL andDermovate Scalp Lotion 0.05% at a 1:1 ratio by volume gave a combinationcontaining BNP at a concentration of 50 μg/mL and clobetasol propionateat a concentration of 250 μg/g. In the present specification, this iscalled a 50 μg/mL BNP:250 μg/g clobetasol combination.

6. Production of CNP:Clobetasol Combination

A combination of CNP and clobetasol propionate was prepared by mixingDermovate (trademark) Scalp Lotion 0.05% (Dermovate Scalp Lotion 0.05%)manufactured by GlaxoSmithKline plc and the CNP gel obtained in ‘1.Production of gel’ above at equal volumes. In the present specification,this is called a CNP:clobetasol combination. Since Dermovate ScalpLotion 0.05% contains clobetasol propionate at a concentration of 500μg/g, mixing the CNP gel having a concentration of 100 μg/mL andDermovate Scalp Lotion 0.05% at a 1:1 ratio by volume gave a combinationcontaining CNP at a concentration of 50 μg/mL and clobetasol propionateat a concentration of 250 μg/g. In the present specification, this iscalled a 50 μg/mL CNP:250 μg/g clobetasol combination.

7. Production of CNP:Carpronium Chloride Combination

A combination of CNP and carpronium chloride was prepared by mixingCalpranin (trademark) solution 5% (Calpranin) manufactured by TaiyoPharmaceutical Industry Co., Ltd. with the CNP gel obtained in ‘1.Production of gel’ above at equal volumes. In the present specification,this is called a CNP:carpronium chloride combination. Since theCalpranin solution 5% contains carpronium chloride at a concentration of50 mg/mL, mixing the CNP gel having a concentration of 100 μg/mL and theCalpranin solution 5% at a 1:1 ratio by volume gave a combinationcontaining CNP at a concentration of 50 μg/mL and carpronium chloride ata concentration of 50 mg/mL. In the present specification, this iscalled a 50 μg/mL CNP gel:50 mg/mL carpronium chloride combination.

Example 3 1. Diagnosis and Treatment of Test Subjects

Prior to administration of the ANP gel, BNP gel, CNP gel, BNP ointment,and CNP ointment of the present invention, as is routine fordermatological diagnosis and treatment, the test subjects wereinterviewed regarding age, gender, history of disease, and familyhistory of disease; when an allergic predisposition was suspected, ascratch test against the main allergens and an evaluation thereof werecarried out. In dermatological diagnosis and treatment, since there area relatively large number of patients who have strong immunoreactivitytoward a specific allergen, or a disease such as atopic dermatitis thatis suspected to be related to having an allergic predisposition, inorder to assist diagnosis, an interview with respect to family historyand previous history of allergic disease in addition to gender and age,and a scratch test toward the main allergens, are widely carried out assimple supplemental test methods.

With regard to evaluation of the results of the scratch test, when areactivity of 1+ or above is exhibited toward any allergen, there can besaid to be a more or less allergic predisposition. In the case ofpatients who come to the dermatology department, many show a reactivityof 1+ or 2+ toward some allergens in the scratch test, and there areonly few patients who do not show any reactivity toward any of theallergens. Since the scratch test is a simple test for estimating thepresence or absence of an allergic predisposition, it cannot preciselyevaluate whether or not there is allergic predisposition but,empirically, if a reactivity of 2+ is shown for a specific allergen, therelationship with an immune disease should be noted when carrying outdiagnosis.

Table 1 to Table 23 show the results of interviews with the testsubjects and the results of the scratch test together with evaluation ofthe diagnostic findings and symptoms of the test subjects carried out in[Example 1] above.

The therapeutic effects of the agent for the treatment of alopecia ofthe present invention on each alopecia are summarized in the Tables asfollows.

Therapeutic Effect on Alopecia Areata:

ANP: Table 1, Table 2

BNP: Table 3-1, Table 3-2

CNP: Table 4, Table 5, Table 6, Table 7-1

BNP:betamethasone:gentamicin combination: Table 7-2

CNP:betamethasone:gentamicin combination: Table 7-3

Therapeutic Effect on Androgenetic Alopecia:

ANP: Table 8

BNP: Table 9-1, Table 9-2

CNP: Table 10-1, Table 10-2

CNP:betamethasone:gentamicin combination: Table 10-3

CNP:clobetasol combination: Table 10-4

CNP:carpronium chloride combination: Table 10-5

Multilayer application of CNP gel and minoxidil: Table 10-6

Multilayer application of BNP gel and minoxidil: Table 10-7

Therapeutic Effect on Postpartum Alopecia:

ANP: Table 10-8

BNP: Table 10-9

CNP: Table 11-1

Therapeutic Effect on Female Pattern Alopecia:

BNP: Table 11-2

CNP: Table 12

Therapeutic Effect on Seborrheic Alopecia:

ANP: Table 13

BNP: Table 14

Therapeutic Effect on Alopecia Pityroides:

ANP: Table 15

BNP: Table 16

CNP: Table 17, Table 18

Therapeutic Effect on Senile Alopecia:

ANP: Table 19

BNP: Table 20

CNP: Table 21

Therapeutic Effect on Cancer Chemotherapy Drug-Induced Alopecia:

BNP: Table 22

CNP: Table 23

The ‘non-recurrence period’ referred to here means the period duringwhich the symptoms did not recur even when treatment with thepreparation of the present invention was discontinued after the symptomshad been relieved. In order to carry out evaluation objectively,photographs before and after application of the NP preparation wererecorded in all cases. Photographs of some of these cases are shown inthe drawings.

Example 4 2. Therapeutic Effect of ANP Gel on Alopecia Areata

The therapeutic effects of the ANP gel on alopecia areata are shown inTable 1 (cases A1 to A5) and Table 2 (cases A6 and A7).

TABLE 1 Case A1 (=C5) A2 A3 (=C15) A4 A5 (=B3) Figure FIG. 7 FIG. 8-1FIG. 9 FIG. 10 FIG. 8-2 Gender Female Female Female Male Male Age 33years old 33 years old 52 years old 32 years old 16 years old Whendeveloped 13 years old 31 years old 51 years old 1 month earlier 7 yearsold Hair loss range S3 S3 S3 S1 S3 Treatment site Right temporal Righttemporal Right temporal Left temporal Right temporal region region, backof region region, crown, region the head, crown right temporal regionHair loss site other Eyebrows (B1) None (B0) None (B0) None (B0)Eyebrows, lower than head limbs (B1) Family history of None None NoneNone None alopecia Family history of Mother: atopic None None None Noneimmune disease dermatitis Older sister: atopic dermatitis, allergicrhinitis Previous history Atopic None Allergic Atopic Atopic dermatitis,rhinitis dermatitis, dermatitis atopic alopecia allergic rhinitisScratch Test House dust: 2+ House dust: 2+ House dust: 1+ House dust: 2+House dust: 3+ Mite: 1+ Mite: 3+ Mite: — Mite: 3+ Mite: 3+ Cedar: —Cedar: 3+ Cedar: 2+ Cedar: 2− Cedar: — Dactylis: 2+ Dactylis: 2+Dactylis: 1+ Dactylis: 2+ Dactylis: 2+ Ragweed: 2+ Ragweed: 2+ Ragweed:1+ Ragweed: 2+ Ragweed: 1+ Effect of minoxidil Not applied Not appliedNot applied Not applied Not applied Effect of steroid drug No effect Noeffect Unknown Not applied No effect Effect of cooling therapy No effectNot applied Not applied Not applied Not applied Antiallergic drug Notapplied Not applied Not applied Not applied Not applied Effect ofcarpronium Not applied No effect No effect Not applied Not appliedchloride Effect of cepharanthin Not applied No effect Not applied Notapplied Not applied Dosage form ANP gel ANP gel ANP gel ANP gel ANP gel(changed to ANP gel after 2 weeks' application of CNP ointment) Dose 100μg/g 100 μg/g 100 μg/g 100 μg/g 100 μg/g Number of days used 21 days 5weeks 5 weeks 4 weeks 21 days Degree of improvement S3→S3 S3→S3 S3→S2S1→S0 S3→S3 in symptoms Non-recurrence period Insufficient InsufficientTreatment 2 weeks No effect effect effect continuingCase A1

The test subject was a 33 year old female and was a patient with severealopecia areata for which the hair loss range was S3 and which, otherthan the head, was accompanied by B1 hair loss of the eyebrows. Therewas a previous history of atopic dermatitis, and coexisting alopecia dueto exacerbation of atopic dermatitis. The results of the scratch testwere house dust 2+, mite 1+, Dactylis 2+, and ragweed 2+. This testsubject showed erythema and pityriatic scale accompanied by itching inthe scalp of the hair loss site, and there was coexisting alopeciapityroides. For this test subject, a circular bald area had appeared atthe age of 13 and also in the crown at the age of 14, and it had thenbecome alopecia totalis in half a month. Following this, the symptoms ofthe test subject had fluctuated, and alopecia had extended to the entirebody at the age of 15. External and orally administered steroid had notgiven any therapeutic effect at all with this test subject. This testsubject had continued to receive stimulation therapy with liquidnitrogen, but there had been hardly any therapeutic effect. This testsubject was the same test subject as that of case C5.

When 100 μg/g ANP gel was applied to the entire bald area on the righttemporal region of this test subject twice a day for 3 weeks, growth ofvellus hair was observed, but erythema and pityriatic desquamation ofthe scalp were not relieved and there was itching.

Case A2

The test subject was a 33 year old female and was a patient with severealopecia areata having a hair loss range of S3. The results of thescratch test were house dust 2+, mite 3+, cedar 3+, Dactylis 2+, andragweed 2+. This test subject had been affected by alopecia areata 2years earlier and had two bald patches, which had cured spontaneously.This time, after stress in the workplace had continued for about 1 monthhalf a year earlier, a bald area had appeared on the right temporalregion, the back of the head, and the crown. This test subject had for 6months been subjected to external steroid treatment, application ofcarpronium chloride, and orally administered cepharanthin, but there hadbeen no change at all.

When 100 μg/g ANP gel was applied to the bald area on the right temporalregion, the back of the head, and the crown of this test subject twice aday, hair growth was clearly confirmed in 1 week, and growth of shortblack hair was observed over a wider area in 2 weeks, but in terms ofsubjective symptoms felt by the test subject there was still a largeamount of hair falling out. The rate of growth of hair after the ANP gelwas applied to this test subject was slow, and there was no formation ofterminal hair. A photograph of the bald area before application and aphotograph of the bald area after 100 μg/g ANP gel was applied twice aday for 5 weeks are shown in FIG. 7. In the case of this test subject,although vellus hair growth was observed, formation of terminal hair wasnot seen, and the hair did not become long.

Case A3

The test subject was a 52 year old female and was a patient with severealopecia areata having a hair loss range of S3. There was a previoushistory of allergic rhinitis. The results of the scratch test were housedust 1+, cedar 2+, Dactylis 1+, and ragweed 1+. A bald patch hadappeared on the frontal region of this test subject about 1 year earlierwhen doing continuous night shifts, following this there had been atendency for enlargement, and multiple bald areas had appeared over theentire scalp. Application of carpronium chloride to the bald patch hadcontinued for 8 months, but the hair loss range had only enlarged andthere had been no therapeutic effect. This test subject was a case inwhich there were no inflammatory symptoms such as erythema, crust, orscale on the scalp of the hair loss area. This test subject was the sametest subject as the test subject of case C15.

When 100 μg/g CNP ointment was applied to the entire bald area of theright temporal region of this test subject twice a day, hair growth wasconfirmed in 1 week, further marked hair growth was observed in 2 weeks,and enlargement of the bald area stopped completely. Application of theCNP ointment was stopped after 14 days, from the next day after that 100μg/g ANP gel was applied twice a day with the expectation of furthereffects, and the therapeutic effect of the CNP ointment continued as itwas; currently, with 5 weeks elapsed after the treatment with CNPointment started, although application of the ANP gel is continuing,thickening of terminal hair and enlargement of the hair growth area canbe seen (Ref. FIG. 8-1 and FIG. 8-2). This test subject could not besubjected to continuing treatment thereafter, and the bald arearemained.

Case A4

The test subject was a 32 year old male and was a patient with alopeciaareata having a hair loss range of S1. There was a previous history ofatopic dermatitis and allergic rhinitis. The results of the scratch testwere house dust 2+, mite 3+, cedar 2+, Dactylis 2+, and ragweed 2+. Abald area, triggered by mental stress, had appeared in the left temporalregion of this test subject 1 month earlier, and following this baldareas had been observed in the crown and the right temporal region. Thiswas accompanied by itching.

When 100 μg/g ANP gel was applied to the entire bald area of this testsubject twice a day, hair growth was confirmed in 1 week on all of theleft temporal region, the crown, and the right temporal region.Following this, there was a cure 4 weeks after starting application.Currently, with 2 weeks elapsed after application of the ANP gel wasstopped after 4 weeks, there is no recurrence of the bald patch (Ref.FIG. 9). However, application of the ANP gel to this test subject didnot grow hair at the hair line on the back of the head.

Case A5

The test subject was a 16 year old male and was a patient with alopeciaophiasis type alopecia areata having a hair loss range of S3, which wassaid to be intractable. This test subject was also affected by atopicdermatitis. The results of the scratch test were house dust 3+, mite 3+,Dactylis 2+, and ragweed 1+. This test subject had been subjected tosurgical removal of the adenoids and palatine tonsils at the age of 6,alopecia areata had occurred from the age of 7, and hair loss was alsoobserved in the eyebrows and lower limbs in addition to the head. Thistest subject had not been cured by internal or external use of asteroid. Invasive erythema and scale accompanied by itching wereobserved on the scalp of the hair loss site of this test subject. Thistest subject was the same test subject as the test subject of case B3.

When 100 μg/g ANP gel was applied to the entire bald area of the righttemporal region of this test subject twice a day for 3 weeks, theerythema seemed to be relieved slightly, but short terminal hairremaining around the hair loss area fell out and the hair loss range wassomewhat enlarged (Ref. T1 of FIG. 10).

TABLE 2 Case A6 (=C3, C4) A7 (=C6) Figure FIG. 11 Gender Female FemaleAge 50 years old 22 years old When developed 40 years old >1.5 yearsearlier Hair loss range S2 S3 Treatment site Crown Frontal region,temporal region ophiasic loss area Hair loss site other None (B0)Eyebrows (B1) than head Family history of Mother: alopecia None alopeciaareata Family history of Child: allergic Grandfather: atopic immunedisease rhinitis, chronic dermatitis urticaria Mother, older brother,older sister: allergic rhinitis Previous history Atopic dermatitis,Atopic dermatitis, allergic rhinitis allergic rhinitis Scratch TestHouse dust: 1+ House dust: 2+ Mite: 1+ Mite: 3+ Cedar: — Cedar: —Dactylis: 2+ Dactylis: 1+ Ragweed: 1+ Ragweed: — Effect of minoxidil Notapplied Not applied Effect of steroid drug No effect No effect Effect ofcooling therapy Not applied Not applied Antiallergic drug No effect Noeffect Effect of carpronium No effect Not applied chloride Effect ofcepharanthin Not applied Not applied Dosage form ANP gel ANP gel Dose100 μg/g 50 μg/g Number of days used 14 days 3 days Degree ofimprovement S2→S2 S3→S3 in symptoms Non-recurrence period No effect NoeffectCase A6

The test subject was a 50 year old female and was a patient with severealopecia areata multilocularis having a hair loss range of S2. There wasa previous history of atopic dermatitis and allergic rhinitis. Themother of the subject had a history of alopecia areata, and a child wasaffected by allergic rhinitis and chronic urticaria. The results of thescratch test were house dust 1+, mite 1+, Dactylis 2+, and ragweed 1+.Erythema and pityriatic scale accompanied by itching were observed onthe scalp of the hair loss site of this test subject, and there wascoexisting alopecia pityroides. This test subject had been affected byalopecia areata multilocularis 10 years earlier, and there had beenrepeated partial remission and recurrence of alopecia areata. When thistest subject became busy at work, the symptoms of alopecia tended todeteriorate. There had been no effect from the external use of steroidand carpronium chloride and an orally administered antiallergy drug for6 months. This test subject was the same test subject as the testsubject of case C3 and case C4.

100 μg/g ANP gel was applied to the multiple bald areas on the crown ofthis test subject for 2 weeks, but there was no effect, rough scaleincreased, erythema appeared, itchiness occurred, the amount of hairfalling out increased, and the hair loss range enlarged somewhat (Ref.FIG. 11).

Case A7

The test subject was a 22 year old female and was a patient with severealopecia ophiasis type alopecia areata having a hair loss range of S3,which was said to be intractable. This test subject had a previoushistory of atopic dermatitis and allergic rhinitis. A grandfather ofthis test subject was affected by atopic dermatitis, and the father, themother, and the brother were affected by allergic rhinitis. The resultsof the scratch test were house dust 2+, mite 3+, and Dactylis 1+. Thistest subject had been affected by a bald area more than one and halfyears earlier, and a band-shaped bald area in a state in which hair didnot grow at all was observed with a clear border along the outside edgeof the part where hair was growing. Erythema, scale, crust, andinflammatory symptoms that seemed to be due to atopic alopecia wereobserved on the hair loss site of this test subject, and wereaccompanied by itching. This test subject had hair loss on the eyebrowsin addition to the head. The skin of this test subject was in anerythrodermic state, which seemed to be due to steroid rebound, andredness and infiltration were observed on the skin of the entire bodyincluding the scalp. There had been no therapeutic effect at all on thistest subject from treatment with a steroid and an antiallergy drug. Thistest subject was the same test subject as the test subject of case C6.

50 μg/g ANP gel was applied to the entire bald area of this test subjecttwice a day for 3 days, but there was no improvement in redness oritching, redness appeared on the scalp somewhat, and there was no signof hair growth.

3. Therapeutic Effect of BNP Gel on Alopecia Areata

The therapeutic effects of BNP gel are shown in Table 3-1 (cases B1 toB4 and B13) and Table 3-2 (case B26).

TABLE 3-1 Case B1 (=C12) B2 B3 B4 (C9) B13 (C13) Figure FIG. 12-1 FIG.13 FIG. 10 FIG. 14 FIG. 34 FIG. 12-2 FIG. 57 Gender Female Male MaleFemale Male Age 63 years old 13 years old 16 years old 24 years old 39years old When developed 62 years old 1 month earlier 7 years old As ajunior high 2 months earlier (1.5 years school student earlier) Hairloss range S2 S1 S3 S2 S1 Treatment site Right temporal Crown Righttemporal Left frontal Right temporal region region region, right region,back of frontal region the head Hair loss site other None (B0) None (B0)Eyebrows, lower None (B0) None (B0) than head limbs (B1) Family historyof None None None None None alopecia Family history of None None NoneMother: None immune disease bronchial asthma Previous history AllergicNone Atopic Allergic None rhinitis, atopic dermatitis rhinitisdermatitis Scratch Test House dust: 2+ Not tested House dust: 3+ Housedust: 2+ House dust: 1+ Mite: 1+ Mite: 3+ Mite: 2+ Mite: 1+ Cedar: —Cedar: — Cedar: 2+ Cedar: 1+ Dactylis: 2+ Dactylis: 2+ Dactylis: 1+Dactylis: — Ragweed: 1+ Ragweed: 1+ Ragweed: 1+ Ragweed: 1+ Effect ofminoxidil Not applied Not applied Not applied Not applied Not appliedEffect of steroid drug No effect Not applied No effect No effect Notapplied Effect of cooling therapy Not applied Not applied Not appliedNot applied Not applied Antiallergic drug Not applied Not applied Notapplied No effect Not applied Effect of carpronium No effect Not appliedNot applied No effect No effect chloride Effect of cepharanthin Notapplied Not applied Not applied Not applied Not applied Dosage form BNPgel BNP gel BNP gel BNP gel (left BNP gel frontal region) Dose 100 μg/g50 μg/g 100 μg/g 100 μg/g 50 μg/g Number of days used 24 days 7 days 14days 21 days 14 days Degree of improvement S2→S0 S1→S0 (Note) S2→S0S1→S0 in symptoms Non-recurrence period 8 months 9 months 4 months 1year 1 year (Note): applied only to right temporal region and not toentire region, degree of improvement could not be evaluated byproportion (S) of bald patch area occupying entire head area.Case B1

The test subject was a 63 year old female and was a patient with severealopecia areata multilocularis having a hair loss range of S2. This testsubject had a history of allergic rhinitis, and there was coexistingatopic dermatitis. The results of the scratch test were house dust 2+,mite 1+, Dactylis 2+, and ragweed 1+. The alopecia areata of this testsubject had continued for over one and a half years. This test subjecthad previously received application of a steroid and carpronium chlorideto the bald area continuously for 10 months, but there had been noeffect, and no hair growth had been observed. This test subject was thesame test subject as the test subject of case C12.

When 100 μg/g BNP gel was applied to the entire bald area of the righttemporal region of this test subject twice a day morning and evening,clear hair growth was observed in 2 weeks. After the application of BNPgel was stopped after 24 days, there was no recurrence of hair loss atthe application site even after 8 months had elapsed (Ref. FIG. 12-1 andFIG. 12-2).

Case B2

The test subject was a 13 year old male with alopecia areatamonolocularis having a hair loss range of S1. This test subject was notsubjected to a scratch test.

When 50 μg/g BNP gel was applied to the entire bald area of this testsubject twice a day for 1 week, hair growth was clearly confirmed andthere was a cure. After application of the BNP gel was stopped after 7days, there was no recurrence even after 9 months had elapsed (Ref. FIG.13).

Case B3

The test subject was a 16 year old male and was a patient with ophiasistype alopecia areata having a hair loss range of S3, which was said tobe intractable. There was coexisting atopic dermatitis. The results ofthe scratch test were house dust 3+, mite 3+, Dactylis 2+, and ragweed1+. This test subject had been subjected to surgical removal of theadenoids and palatine tonsils at the age of 6, alopecia areata hadoccurred from the age of 7, and hair loss was also observed in theeyebrows and lower limbs in addition to the head. This test subject hadnot been cured by internal or external use of a steroid. Invasiveerythema and scale accompanied by itching were observed on the scalp ofthe hair loss site of this test subject. This test subject was the sametest subject as the test subject of case A5.

When 100 μg/g ANP gel was applied to the bald area of the right temporalregion of this test subject twice a day for 3 weeks, erythema seemed tobe relieved slightly, but short terminal hair remaining around the hairloss area fell out and the hair loss range enlarged somewhat. When 100μg/g BNP gel was then applied to the hair loss area of the righttemporal region twice a day from 2 days after the ANP gel was stopped,marked hair growth was observed in 2 weeks (Ref. T2 of FIG. 10). Therewas no hair growth at all in the left temporal region, to which the BNPwas not applied. After application of the BNP gel was stopped after 14days, there was no recurrence for 4 months, but a new bald area occurredin another place at the time of a school entrance examination when 4months had elapsed.

This test subject did not come to the clinic thereafter, and theprogress thereafter is not known.

Case B4

The test subject was a 24 year old female and was a patient with severealopecia areata multilocularis having a hair loss range of S2. There wasa history of allergic rhinitis. The mother was affected by bronchialasthma. The results of the scratch test were house dust 2+, mite 2+,cedar 2+, Dactylis 1+, and ragweed 1+. There had been repeated partialremission and recurrence of the alopecia areata of this test subjectsince junior high school. A slight degree of itching sensation hadstarted to appear on and around the frontal region half a year earlier,and following this the multiple bald areas enlarged. Even when a steroidor carpronium chloride had been applied to the hair loss site of thistest subject for 3 months or an antiallergy drug had been orallyadministered to the test subject, hair growth had not been observed, andthe susceptibility to hair loss could not be improved. This test subjectwas the same test subject as the test subject of case C9.

In accordance with the separate left and right application method, 100μg/g BNP gel was applied to the left frontal region of this testsubject, and only Lubrajel NP (ISP Japan Ltd.), which is a gel base, wasapplied to the right frontal region twice a day; hair growth wasobserved after 1 week only on the site of the left frontal region towhich the BNP gel was applied, and after 2 weeks hair growth became moremarked on the site of the left frontal region to which the BNP gel hadbeen applied. On the other hand, no hair growth was observed on theright frontal region to which only the gel base had been applied.Therefore, it was determined that the hair growth was due to the hairgrowth effect of the BNP gel. Application of 100 μg/g BNP gel to theleft frontal region was continued, application of 100 μg/g CNP ointmentto the right frontal region was started, hair growth was also observedon the right frontal region after 1 week, and marked hair growth andhair thickening were observed on the entire hair loss site of the rightfrontal region after 3 weeks. Before starting the treatment two handfulsof hair had fallen out each time it was shampooed, but the amount ofhair falling out decreased dramatically, and only 5 to 6 hairs fell outper shampooing. External application was stopped after the BNP gel hadbeen applied to the left frontal region for 3 weeks and the CNP ointmentto the right frontal region for 3 weeks, but following that haircontinued to grow on the left frontal region and the right frontalregion, the alopecia was substantially cured by the second week afterapplication was stopped, and subsequently there was a complete cure. Forthis test subject, there has been no recurrence up to the present, thatis, after a further 1 year has elapsed (FIG. 14 and FIG. 57).

Case B13

The test subject was a 39 year old male and is the same test subject asthe test subject of case C13 described later. When 100 μg/g CNP ointmentwas applied twice a day to a circular bald area of the temporal regionand the back of the head of this test subject, hair grew after 1 week'sapplication, and it spontaneously cured after the application wasstopped at 1 week. However, a new circular bald area occurred on thecrown after 10 months had elapsed since application of the CNP ointmenthad stopped.

When 50 μg/g BNP gel was applied twice a day to the circular bald areaof the crown of this test subject, growth of black terminal hair wasobserved on the entire bald area of the crown after 2 weeks' application(FIG. 34).

Progress was examined after application of the BNP gel was stopped at 2weeks, but since hair growth was not observed in a central area, a 50μg/g BNP:600 μg/mL betamethasone:500 μg/mL gentamicin combination wasapplied once a day for 2 weeks starting 3 weeks after the BNP gel wasstopped, and black terminal hair grew at a higher hair growth rate.

4. Therapeutic Effect of BNP Ointment on Alopecia Areata

The therapeutic effects of BNP ointment are shown in Table 3-2 (testsubjects B5 and B6).

TABLE 3-2 Case B5 B6 (C10) B26 (C7) Figure FIG. 15-1 FIG. 55 FIG. 15-2FIG. 16 Gender Female Female Female Age 50 years old 33 years old 38years old When developed 4 months earlier 5 months earlier 1 monthearlier Hair loss range S3 S1 S1 Treatment site Crown, Temporal regionLeft temporal region, Back of the head crown, back of the head Hair losssite other None (B0) None (B0) None (B0) than head Family history ofNone None Relative: alopecia alopecia areata Family history of NoneChild: atopic dermatitis Child: atopic dermatitis immune diseasePrevious history Allergic rhinitis, atopic None None dermatitis ScratchTest House dust: 3+ House dust: 2+ Not tested Mite: 3+ Mite: 2+ Cedar:3+ Cedar: — Dactylis: 2+ Dactylis: 2+ Ragweed: 2+ Ragweed: 2+ Effect ofminoxidil Not applied Not applied Not applied Effect of steroid drug Noeffect No effect No effect Effect of cooling therapy Not applied Notapplied Not applied Antiallergic drug Not applied No effect No effectEffect of carpronium No effect No effect No effect chloride Effect ofcepharanthin Not applied Not applied Not applied Dosage form BNPointment BNP ointment BNP gel Dose 30 μg/g 100 μg/g 50 μg/g 50 μg/gNumber of days used 35 days 7 days 14 days Degree of improvement S3→S0S1→S0 S1→S0 in symptoms Non-recurrence period Immediately after end of 1month 3 weeks treatmentCase B5

The test subject was a 50 year old female and was a patient with severealopecia areata multilocularis having a hair loss range of S3. There wasa history of allergic rhinitis, and there was coexisting atopicdermatitis. The results of the scratch test were house dust 3+, mite 3+,cedar 3+, Dactylis 2+, and ragweed 2+. Alopecia areata of this testsubject had developed 4 months earlier due to stress caused byrelationships in the workplace, and 2 months after the onsetmultilocularis started. Steroid lotion and carpronium chloride had beenapplied to the hair loss site of this test subject continuously for 10months, but no hair growth had been observed, susceptibility to hairloss had not been suppressed, and there had been no therapeutic effects.

When 50 μg/g BNP ointment was applied to the entire bald area of thecrown and temporal region of this test subject twice a day morning andevening, hair completely stopped falling out after starting theapplication. When application of the BNP ointment to the entire hairloss site was continued for 2 weeks, hair growth could be clearlyconfirmed. Application of 50 μg/g BNP ointment twice a day was endedafter 2 weeks, and when from the next day after that 30 μg/g BNPointment was applied twice a day for 1 week, and from the next day afterthat 50 μg/g BNP ointment was applied twice a day for 2 weeks, hairthickening continued to progress. The hair growth had a characteristicpattern, hair started to grow in double rings and recovered so as togive complete coverage in 20 days (Ref. FIG. 15-1, FIG. 15-2, and FIG.16). However, since this test subject was affected by the Great EastJapan Earthquake on 11 Mar. 2011, treatment with the BNP ointment wasstopped. According to this test subject, the hair grew to some extent,but it stopped growing after application of the BNP ointment wasstopped.

Case B6

The test subject was a 33 year old female and was a patient withalopecia areata having a hair loss range of S1. A child of this testsubject was affected by atopic dermatitis. The mother of this testsubject had alopecia areata and a history of allergic rhinitis. Theresults of the scratch test were house dust 2+, mite 2+, Dactylis 2+,and ragweed 2+, which were those of a case with an atopicpredisposition. This test subject had developed a circular bald area 5months earlier, treatments involving external use of a steroid,application of carpronium chloride, and an orally administeredantiallergy drug had been continued for 7 weeks, but not only had haircontinued to fall out but the hair loss range had also enlarged andmultilocularis was seen; orally administered steroid had been given incombination, but there had been no therapeutic effect. This test subjectshowed bald areas on the left temporal region, the crown, and the backof the head, with a hair loss range of S1. This test subject was thesame test subject as the test subject of case C10.

Among the bald areas of this test subject, when 100 μg/g BNP ointmentwas applied to the left temporal region and the crown twice a day,growth of mainly white hair was observed on the 7^(th) day after theapplication of BNP ointment was started. On the other hand, hair growthwas not observed on the bald area of the back of the head, to which theBNP ointment had not been applied. However, since there was still alarge amount of hair falling out, application of the BNP ointment wasstopped after 7 days, and 100 μg/g CNP ointment was applied twice a dayto all the bald areas of the left temporal region, the crown, and theback of the head from the 8^(th) day. As a result, hair stopped fallingout, marked hair growth was confirmed on all the bald areas includingthe back of the head after 3 weeks, and there was a cure.

Case B26

The test subject was a 38 year old female and was a patient withalopecia areata monolocularis having a hair loss range of S1. A child ofthis test subject was affected by atopic dermatitis. A cousin, a niece,an aunt, and a child of this test subject were affected by alopeciaareata multilocularis. This test subject was not subjected to a scratchtest.

When 100 μg/g CNP ointment was applied twice a day for 2 weeks to theentire circular bald area of the crown of this test subject, clear hairgrowth was observed (Ref. FIG. 23). There was no recurrence of the baldarea in the site that had been cured by the CNP ointment even when 1year had elapsed since application of the CNP ointment was stopped after2 weeks.

However, one year after the circular bald area on the crown had beencured by the CNP ointment a new circular bald area appeared, this timeon the back of the head.

When 50 μg/g BNP gel was applied twice a day for 2 weeks, hair grewmarkedly and became terminal hair, and there was almost a cure (FIG.55). Application of the BNP gel was stopped after 2 weeks, and there wasa complete cure and no recurrence even when 6 weeks had subsequentlyelapsed (FIG. 55).

This test subject was the same test subject as the test subject of caseC7.

5. Therapeutic Effect of CNP Gel on Alopecia Areata

The therapeutic effects of CNP gel on alopecia areata are shown in Table4 (test subjects C1, C3, C20, and C23).

TABLE 4 Case C1 C2 (=C11) C3 (=A6, C4) C20 Figure FIG. 17 FIG. 18-1 FIG.20 FIG. 35 FIG. 18-2 FIG. 19-1 FIG. 19-2 Gender Female Female FemaleMale Age 32 years old 47 years old 50 years old 32 years old Whendeveloped Half year earlier 1 month earlier 40 years old About 30 yearsold Hair loss range S2 S1 S2 Frontal region Treatment site Crown Crown,frontal Left temporal Hairline of the region, left region frontal regiontemporal region became thin. Circular bald area seen in left temporalregion. Hair loss site other None (B0) None (B0) None (B0) None thanhead Family history of None None Mother: alopecia None alopecia areataFamily history of None Child: allergic Child: allergic None immunedisease rhinitis rhinitis, chronic urticaria Previous history NoneGlaucoma Atopic dermatitis, Not tested allergic rhinitis Scratch TestHouse dust: 2+ House dust: 3+ House dust: 1+ Not applied Mite: 3+ Mite:3+ Mite: 1+ Cedar: 2+ Cedar: 2+ Cedar: — Dactylis: 3+ Dactylis: 2+Dactylis: 2+ Ragweed: 2+ Ragweed: 2+ Ragweed: 1+ Effect of minoxidil Notapplied Not applied Not applied Not applied Effect of steroid drug Notapplied Unable to use No effect Not applied Effect of cooling therapyNot applied Not applied Not applied Not applied Antiallergic drug Notapplied Not applied No effect Not applied Effect of carpronium No effectNot applied No effect Not applied chloride Effect of cepharanthin Notapplied Not applied Not applied Not applied Dosage form CNP gel CNPointment CNP gel CNP gel Dose 100 μg/g 100 μg/g 100 μg/g 50 μg/ml Numberof days used 7 days 28 days 21 days 14 days Degree of improvement S2→S2S1→S0 S2→S0 S1→S0 in symptoms Non-recurrence period Unknown 3 weeks 8months 5 weeksCase C1

The test subject was a 32 year old female and was a patient with aserious case of alopecia areata multilocularis having a hair loss rangeof S3. There was a history of allergic rhinitis. The results of thescratch test were house dust 2+, mite 3+, cedar 2+, Dactylis 3+, andragweed 2+. With regard to the alopecia areata of this test subject,alopecia had developed half a year earlier, it had enlarged, and therewere multiple occurrences, and a tendency for hair to be lost over theentire scalp had been seen 5 months after onset. For this test subject,a change of workplace had triggered the onset of alopecia areata. Thistest subject had not shown any therapeutic effect from the applicationof carpronium chloride.

When 100 μg/g CNP gel was applied twice a day to the entire bald area ofthe crown of this test subject, marked hair growth was observed after 1week (Ref. FIG. 17).

Case C2

The test subject was a 47 year old female and was a patient withalopecia areata having a hair loss range of S1. This test subject had aprevious history of glaucoma. A child of this test subject was affectedby atopic dermatitis and allergic rhinitis. The results of the scratchtest were house dust 3+, mite 3+, cedar 2+, Dactylis 2+, and ragweed 2+.A bald patch had appeared on the crown of this test subject 1 monthearlier, and following this bald areas had been observed on the frontalregion and left temporal region. Since this test subject had a historyof glaucoma, this was a case in which use of a steroid should beavoided. This test subject had been affected by thyroid cancer 7 yearsearlier and had received surgical removal. This test subject was thesame test subject as the test subject of case C11.

When 100 μg/g CNP ointment was applied to the crown and the frontalregion of this test subject twice a day, hair growth was confirmed after1 week of application (Ref. FIG. 18-1, FIG. 19-1, and FIG. 19-2). Fromthe following day, that is, the 8^(th) day, the dosage form was changedon request from the test subject, 100 μg/g CNP gel continued to beapplied twice a day for a further 1 week, further marked hair growth wasobserved, and the amount of hair falling out decreased (Ref. FIG. 18-1,FIG. 18-2, FIG. 19-1, and FIG. 19-2). However, in the left temporalregion of this test subject, to which external CNP ointment had not beenapplied, there was no hair growth. Since an effect had been confirmed,from the 8^(th) day after application of the CNP ointment to the crownand the frontal region was started, application of 100 μg/g CNP gel tothe left temporal region twice a day was started, and 2 weeks after thestart of application marked hair growth was confirmed for the lefttemporal region in the same way as for the crown and the frontal region.There was no recurrence even when 3 weeks had elapsed after applicationof the CNP gel was stopped after 28 days.

Case C3

The test subject was a 50 year old female and was a patient with aserious case of alopecia areata multilocularis having a hair loss rangeof S2. This test subject had a previous history of atopic dermatitis andallergic rhinitis. The mother of this test subject had a history ofalopecia areata, and a child was affected by allergic rhinitis andchronic urticaria. The results of the scratch test were house dust 1+,mite 1+, Dactylis 2+, and ragweed 1+. This test subject showed erythemaand pityriatic scale accompanied by itching on the scalp of the hairloss site, and there was coexisting alopecia pityroides. This testsubject had been affected by alopecia areata multilocularis 10 yearsearlier, and there had been repeated partial remission and recurrence.When work became busy, in the case of this test subject the alopeciaareata tended to deteriorate. There had been no effect from external useof a steroid, external use of carpronium chloride, and an orallyadministered antiallergy drug for 6 months. This test subject was thesame test subject as the test subject of case A6 and case C4.

When 100 μg/g CNP gel was applied to the entire bald area of the lefttemporal region of this test subject twice a day, the amount of hairfalling out decreased from the next day, hair growth was clearlyconfirmed after 2 weeks, and application was stopped after 3 weeks'application, but recovery progressed well after the application wasstopped, and there was no recurrence on the application site after 8months had elapsed since stopping the application (Ref. FIG. 20). Therewas no subsequent recurrence for this test subject even when a further 1year had elapsed.

Case C20

The test subject was a 32 year old male with androgenetic alopecia. Forthis test subject, at the age of 30 the hair line of the M-shaped partin the frontal region had rapidly become thin and retreated. Alopeciaareata monolocularis had also developed in the left temporal region 6weeks before visiting the clinic. This test subject had no familyhistory of alopecia patient. This test subject had so-called M-shapedandrogenetic alopecia that was classified as type III on theHamilton-Norwood scale. This test subject was the same test subject asthe test subject of case C27.

When 50 μg/g CNP gel was applied twice a day to the androgeneticalopecia area of the frontal region and the circular bald area of theleft temporal region of this test subject, growth of black terminal hairwas observed 2 weeks later both in the M-shaped hair line area (FIG. 46)and the circular bald area (FIG. 35).

Furthermore, when 50 μg/mL CNP gel:50 mg/mL carpronium chloridecombination was applied twice a day for 1 week to the androgeneticalopecia area of the frontal region and the circular bald area of theleft temporal region of this test subject, the same degree of hairgrowth effect as that from the application of 50 μg/g CNP gel wasmaintained.

Subsequently, when the 50 μg/g CNP:600 μg/mL betamethasone:500 μg/mLgentamicin combination was applied for 1 week after the application of50 μg/g CNP gel was suspended for 3 weeks, further marked hair growthwas observed, and following this even though application was stoppedhair growth continued and there was a cure.

6. Therapeutic Effect of CNP Ointment on Alopecia Areata

The therapeutic effects of CNP ointment on alopecia areata are shown inTable 4 (case C2), Table 5 (test subjects C4 to C7), Table 6 (testsubjects C8 to C12), and Table 7-1 (test subjects C13 to C15, and C36).

TABLE 5 Case C4 (=A6, C3) C5 (=A1) C6 (=A7) C7 Figure FIG. 21 FIG. 22FIG. 23 Gender Female Female Female Female Age 50 years old 33 years old22 years old 38 years old When developed 40 years old 13 years old >1.5years earlier About 1 year earlier Hair loss range S2 S3 S3 S1 Treatmentsite Right frontal Left temporal Frontal region, Crown region regiontemporal region Hair loss site other None (B0) Eyebrows (B1) Eyebrows(B1) None (B0) than head Family history of Mother: alopecia None NoneRelative: alopecia alopecia areata areata Family history of Child:allergic Mother: atopic Grandfather: atopic Child: atopic immune diseaserhinitis, chronic dermatitis dermatitis dermatitis, urticaria Oldersister: Mother, older alopecia areata atopic dermatitis, brother, oldermultilocularis allergic rhinitis sister: allergic rhinitis Previoushistory Atopic dermatitis, Atopic dermatitis Atopic dermatitis, Noneallergic rhinitis allergic rhinitis Scratch Test House dust: 1+ Housedust: 2+ House dust: 2+ Not tested Mite: 1+ Mite: 1+ Mite: 3+ Cedar: —Cedar: — Cedar: — Dactylis: 2+ Dactylis: 2+ Dactylis: 1+ Ragweed: 1+Ragweed: 2+ Ragweed: — Effect of minoxidil Not applied Not applied Notapplied Not applied Effect of steroid drug No effect No effect No effectNo effect Effect of cooling therapy Not applied No effect Not appliedNot applied Antiallergic drug No effect No effect No effect No effectEffect of carpronium No effect Not applied Not applied No effectchloride Effect of cepharanthin Not applied Not applied Not applied Notapplied Dosage form CNP ointment CNP ointment CNP ointment CNP ointmentDose 100 μg/g 100 μg/g 50 μg/g 100 μg/g Number of days used 14 days 21days 28 days 14 days Degree of improvement S2→S1 S3→S1 S3→S0 S1→S0 insymptoms Non-recurrence period Treatment At least 5 months 9 months 1year continuingCase C4

The test subject was a 50 year old female and was a patient with severealopecia areata multilocularis having a hair loss range of S2. This testsubject had a previous history of atopic dermatitis and allergicrhinitis. The mother of this test subject had a history of alopeciaareata, and a child was affected by allergic rhinitis and chronicurticaria. The results of the scratch test were house dust 1+, mite 1+,Dactylis 2+, and ragweed 1+. Erythema and pityriatic scale accompaniedby itching were observed on the scalp of the hair loss site of this testsubject, and there was coexisting alopecia pityroides. This test subjecthad been affected by alopecia areata multilocularis 10 years earlier,and there had been repeated partial remission and recurrence. When workbecame busy, in the case of this test subject the alopecia areata tendedto deteriorate. There had been no effect from external use of a steroid,external use of carpronium chloride, and an orally administeredantiallergy drug for 6 months. This test subject was the same testsubject as the test subject of case A6 and case C3.

When 100 μg/g CNP gel was applied to the entire bald area of the lefttemporal region of this test subject twice a day, the amount of hairfalling out decreased from the next day, hair growth was clearlyconfirmed after 2 weeks, and application was stopped after 3 weeks'application, but recovery progressed well after the application wasstopped, and there was no recurrence on the application site after 8months had elapsed since stopping the application (Ref. FIG. 20).

However, half a year after stopping the application a new bald areaappeared, this time on the right frontal region; 100 μg/g CNP ointmentwas applied twice a day, hair growth was clearly observed in 2 weeks,and erythema and scale of the scalp disappeared. The application to thebald area of the right frontal region was stopped after 2 weeks, but thehair continued to recover even after the application was stopped, andthere was a complete cure. There was no recurrence in the bald area ofthe right frontal region even when over 1 year had elapsed since theapplication was stopped.

100 μg/g ANP gel was applied for 2 weeks to new multiple bald areas onthe crown, which appeared around the same time as the bald area on theright frontal region, but there was no effect, rough scale increased,there was itching, and the hair loss range enlarged somewhat.

Case C5

The test subject was a 33 year old female and was a patient with severealopecia areata for which the hair loss range was S3 and which, otherthan the head, was accompanied by B1 hair loss of the eyebrows. Therewas a previous history of atopic dermatitis, and coexisting alopecia dueto exacerbation of atopic dermatitis. The results of the scratch testwere house dust 2+, mite 1+, Dactylis 2+, and ragweed 2+. This testsubject showed erythema and pityriatic scale accompanied by itching inthe scalp of the hair loss site, and there was coexisting alopeciapityroides. For this test subject, a circular bald area had appeared atthe age of 13 and also in the crown at the age of 14, and it had thenbecome alopecia totalis in half a month. Following this, the symptoms ofthe test subject had fluctuated, and alopecia had extended to the entirebody at the age of 15. External and orally administered steroid had notgiven any therapeutic effect at all with this test subject. This testsubject had continued to receive stimulation therapy with liquidnitrogen, but there had been hardly any therapeutic effect. This testsubject was the same test subject as that of case A1.

When 100 μg/g ANP gel was applied to the bald area on the left temporalregion twice a day for 3 weeks, growth of vellus hair was observed, buterythema and pityriatic desquamation of the scalp were not relieved andthere was itching. Therefore, 100 μg/g CNP ointment was applied to theentire bald area of the left temporal region twice a day for 1 weekstarting 2 weeks after stopping the ANP gel, erythema, pityriaticdesquamation, and the itching sensation of the scalp disappeared, hairrestoration and hair growth were promoted, and terminal hair covered thescalp. Following this, the CNP ointment was applied for another week,and as a result the terminal hair grew and there was almost a cure. 7months have elapsed since application of the CNP ointment was stoppedafter 21 days, but there has been no recurrence of the alopecia (Ref.FIG. 21). This test subject had been subjected to orally administeredsteroid, liquid nitrogen cooling therapy, etc. since her teens, and thiswas the first time that there had been such hair growth.

Case C6

The test subject was a 22 year old female and was a patient with severeophiasis type alopecia areata having a hair loss range of S3, which wassaid to be intractable. This test subject had a previous history ofatopic dermatitis and allergic rhinitis. A grandfather of this testsubject was affected by atopic dermatitis, and the father, the mother,and the brother were affected by allergic rhinitis. The results of thescratch test were house dust 2+, mite 3+, and Dactylis 1+. This testsubject had been affected by a bald area more than one and half yearsearlier, and a band-shaped bald area in a state in which hair did notgrow at all was observed with a clear border along the outside edge ofthe part where hair was growing. Erythema, scale, crust, andinflammatory symptoms that seemed to be due to atopic alopecia wereobserved on the hair loss site of this test subject, and wereaccompanied by itching. This test subject had hair loss on the eyebrowsin addition to the head. This test subject was a case of coexistingatopic dermatitis and allergic rhinitis. The skin of this test subjectwas in an erythrodermic state, which seemed to be due to steroidrebound, and redness and infiltration were observed on the skin of theentire body including the scalp. There had been no therapeutic effect atall on this test subject from treatment with a steroid and anantiallergy drug. This test subject was the same test subject as thetest subject of case A7.

Although 50 μg/g ANP gel was applied twice a day for 3 days to theentire bald area of this test subject, there was no improvement ineither redness or itching, and there was no sign of hair growth.

When it was changed to application of 50 μg/g CNP ointment twice a dayto the entire bald area of the frontal region and the temporal region ofthis test subject, erythema was relieved on the second day, and theitching sensation disappeared. Marked growth of terminal hair wasobserved after 3 weeks' application, and once terminal hair grew, thehair thickened while vellus hair became dark and grew even withoutexternal application, terminal hair also grew, and there was a cure(Ref. FIG. 22). The hair continued to grow even after application of theCNP ointment was stopped after 4 weeks and there was a complete cure.There was no recurrence for this test subject even after 9 months hadelapsed, and the alopecia areata ophiasis was completely cured withoutany marking.

Case C7

The test subject was a 38 year old female and was a patient withalopecia areata monolocularis having a hair loss range of S1. A child ofthis test subject was affected by atopic dermatitis. A cousin, a niece,an aunt, and a child of this test subject were affected by alopeciaareata multilocularis. This test subject was not subjected to a scratchtest.

When 100 μg/g CNP ointment was applied to the entire circular bald areaof the crown of this test subject twice a day for 2 weeks, clear hairgrowth was observed (Ref. FIG. 23). After application of the CNPointment was stopped after 2 weeks, even after 1 year had elapsed therewas no recurrence in the site that had been cured by the CNP ointment.

However, 1 year after the circular bald area of the crown was cured bythe CNP ointment, a new circular bald area appeared, this time on theback of the head.

When 50 μg/g BNP gel was applied twice a day for 2 weeks, there was amarked hair growth and terminal hair formed, and the bald area wasalmost cured.

This test subject was the same test subject as the test subject of caseB26.

TABLE 6 Case C8 C9 (=B4) C10 (=B6) C11 (=C2) C12 (=B1) Figure FIG. 24-1FIG. 18 FIG. 25 FIG. 24-2 Gender Female Female Female Female Female Age32 years old 24 years old 33 years old 47 years old 63 years old Whendeveloped 1.5 years As a junior high 5 months 1 month 64 years oldearlier school student earlier earlier (1.5 years earlier) Hair lossrange S1 S2 S1 S1 S2 Treatment site Right temporal Right frontal Lefttemporal Crown, frontal Left temporal region region region, crown,region region back of the head Hair loss site other None (B0) None (B0)None (B0) None (B0) None (B0) than head Family history of None None NoneNone None alopecia Family history of Child: allergic Mother: bronchialChild: atopic Child: allergic None? immune disease rhinitis asthmadermatitis rhinitis Previous history Allergic Allergic None GlaucomaAllergic rhinitis rhinitis rhinitis, atopic dermatitis Scratch TestHouse dust: — House dust: 2+ House dust: 2+ House dust: 3+ House dust:2+ Mite: 1+ Mite: 2+ Mite: 2+ Mite: 3+ Mite: 1+ Cedar: 2+ Cedar: 2+Cedar: — Cedar: 2+ Cedar: — Dactylis: 2+ Dactylis: 1+ Dactylis: 2+Dactylis: 2+ Dactylis: 2+ Ragweed: 2+ Ragweed: 1+ Ragweed: 2+ Ragweed:2+ Ragweed: 1+ Effect of minoxidil Not applied Not applied Not appliedNot applied Not applied Effect of steroid drug No effect No effect Noeffect Unable to use No effect Effect of cooling therapy Not applied Notapplied Not applied Not applied Not applied Antiallergic drug No effectNo effect No effect Not applied Not applied Effect of carpronium Noeffect No effect No effect Not applied No effect chloride Effect ofcepharanthin Not applied Not applied Not applied Not applied Not appliedDosage form CNP ointment CNP ointment CNP ointment CNP ointment CNPointment (right frontal region) Dose 100 μg/g 100 μg/g 100 μg/g 100 μg/g100 μg/g Number of days used 7 days 21 days 21 days 28 days 20 daysDegree of improvement S1→S0 S2→S0 S1→S0 S1→S0 S2→S0 in symptomsNon-recurrence period 1 month 1 year 1 month 3 weeks 11 monthsCase C8

The test subject was a 32 year old female and was a patient withalopecia areata multilocularis having a hair loss range of S1. Theresults of the scratch test were house dust −, mite 1+, cedar 2+,Dactylis 2+, and ragweed 2+. This test subject had been affected byalopecia multilocularis one and a half years earlier, and had receivedtreatment involving external use of a steroid, application of carproniumchloride, and an orally administered antiallergy drug for one and a halfyears, but a satisfactory effect could not be obtained. A slight degreeof erythema was observed on a hair loss area of this test subject, andwas accompanied by an itching sensation. In the case of this testsubject, multiple bald areas were observed on the right temporal regionand the left temporal region.

When 100 μg/g CNP ointment was applied to the entire bald area of thistest subject twice a day, erythema and the itching sensation disappearedafter 3 days' application, the amount of hair falling out decreasedafter 10 days' application, and clear hair growth was observed. Therewas no recurrence even when 1 month had elapsed after application of theCNP ointment was stopped after 7 days.

Case C9

The test subject was a 24 year old female and was a patient with severealopecia areata multilocularis having a hair loss range of S2. There wasa history of allergic rhinitis. The mother was affected by bronchialasthma. The results of the scratch test were house dust 2+, mite 2+,cedar 2+, Dactylis 1+, and ragweed 1+. There had been repeated partialremission and recurrence of the alopecia areata of this test subjectsince junior high school. A slight degree of itching sensation hadstarted to appear on and around the frontal region half a year earlier,and following this multiple bald areas enlarged. Even when a steroid orcarpronium chloride had been applied to the hair loss site of this testsubject for 3 months or an antiallergy drug had been orally administeredto the test subject, hair growth had not been observed, and thesusceptibility to hair loss could not be improved. This test subject wasthe same test subject as the test subject of case B4.

In accordance with the separate left and right application method, 100μg/g BNP gel was applied to the left frontal region of this testsubject, and only Lubrajel NP (ISP Japan Ltd.), which is a gel base, wasapplied to the right frontal region twice a day; hair growth wasobserved after 1 week only on the site of the left-hand side to whichthe BNP gel had been applied, and after 2 weeks hair growth became moremarked on the site of the left-hand side to which the BNP gel had beenapplied. On the other hand, no hair growth was observed on theright-hand side to which only the gel base had been applied. Therefore,it was determined that the hair growth was due to the hair growth effectof the BNP gel.

Application of 100 μg/g BNP gel to the left frontal region wascontinued, and application of 100 μg/g CNP ointment to the right frontalregion was started, hair growth was observed after 1 week, and markedhair growth and hair thickening were observed on the entire hair losssite after 3 weeks. Before starting the treatment two handfuls of hairhad fallen out each time it was shampooed, but the amount of hairfalling out decreased dramatically, and only 5 to 6 hairs fell out pershampooing. External application was stopped after the BNP gel had beenapplied to the left frontal region for 3 weeks and the CNP ointment tothe right frontal region for 3 weeks, but following this hair continuedto grow, the alopecia was substantially cured by the second week afterapplication was stopped, and there has been no recurrence up to thepresent, that is, after 1 year has elapsed.

Case C10

The test subject was a 33 year old female and was a patient withalopecia areata having a hair loss range of S1. A child of this testsubject was affected by atopic dermatitis. The mother of this testsubject had alopecia areata and a history of allergic rhinitis. Theresults of the scratch test were house dust 2+, mite 2+, Dactylis 2+,and ragweed 2+, which were those of a case with an atopicpredisposition. This test subject had developed a circular bald area 5months earlier, treatments involving the external use of a steroid,application of carpronium chloride, and an orally administeredantiallergy drug had been continued for 7 weeks, but not only had haircontinued to fall out but the hair loss range had also enlarged andmultilocularis was seen; orally administered steroid had been given incombination, but there had been no therapeutic effect. This test subjectshowed bald areas on the left temporal region, the crown, and the backof the head, with a hair loss range of S1. This test subject was thesame test subject as the test subject of case B6.

Among the bald areas of this test subject, when 100 μg/g BNP ointmentwas applied to the left temporal region and the crown twice a day,growth of mainly white hair was observed on the 7^(th) day afterapplication of the BNP ointment was started. On the other hand, hairgrowth was not observed on the bald area of the back of the head, towhich the BNP ointment had not been applied. However, since there wasstill a large amount of hair falling out, application of the BNPointment was stopped after 7 days, and 100 μg/g CNP ointment was appliedtwice a day to all the bald areas of the left temporal region, thecrown, and the back of the head from the 8^(th) day. As a result, hairstopped falling out, and marked hair growth was observed on all of theleft temporal region, the crown, and the back of the head on the 3rdweek after application of the CNP ointment was started (Ref. FIG. 24-1and FIG. 24-2). The test subject was surprised with the rate of hairgrowth compared with conventional therapy. There was no recurrence inthis test subject even when 1 month had elapsed after application of theCNP ointment was stopped after 21 days.

Case C11

The test subject was a 47 year old female and was a patient withalopecia areata having a hair loss range of S1. This test subject had aprevious history of glaucoma. A child of this test subject was affectedby atopic dermatitis and allergic rhinitis. The results of the scratchtest were house dust 3+, mite 3+, cedar 2+, Dactylis 2+, and ragweed 2+.A bald patch had appeared on the crown of this test subject 1 monthearlier, and following this bald areas had been observed on the frontalregion and left temporal region. Since this test subject had a historyof glaucoma, this was a case in which use of a steroid should beavoided. This test subject had been affected by thyroid cancer 7 yearsearlier and had received surgical removal. This test subject was thesame test subject as the test subject of case C2.

When 100 μg/g CNP ointment was applied to the crown and the frontalregion of this test subject twice a day, hair growth was confirmed after1 week of application (Ref. FIG. 18-1, FIG. 18-2, FIG. 19-1 and FIG.19-2).

TABLE 7-1 Case C13 C14 C15 (=A3) C36 C23 Figure FIG. 26 FIG. 8 FIG. 39Gender Male Female Female Male Female Age 38 years old 35 years old 52years old 57 years old 26 years old When developed 2 months 3 months 51years old 55 years old 26 years old earlier earlier Hair loss range S1S2 S3 S2 S1 Treatment site Temporal region, Back of the head Whole ofscalp Temporal region, Back of the head back of the head back of thehead, frontal region Hair loss site other None (B0) None (B0) None (B0)Eyebrows (B1) None than head Family history of None None None None Nonealopecia Family history of None Mother: allergic None None None immunedisease rhinitis Previous history None Allergic rhinitis Allergicrhinitis Atopic dermatitis None Scratch Test House dust: 1+ House dust:2+ House dust: 1+ House dust: 1+ Not tested Mite: 1+ Mite: 2+ Mite: —Mite: 3+ Cedar: 1+ Cedar: 2+ Cedar: 2+ Cedar: 2+ Dactylis: — Dactylis:2+ Dactylis: 1+ Dactylis: 1+ Ragweed: 1+ Ragweed: 1+ Ragweed: 1+Ragweed: 1+ Effect of minoxidil Not applied Not applied Not applied Notapplied Not applied Effect of steroid drug Not applied Hair loss rangeUnknown No effect from external Small amount of enlarged application andoral hair growth, but administration bald area enlarged Effect ofcooling therapy Not applied Not applied Not applied No effect Notapplied Antiallergic drug Not applied Not applied Not applied No effectNot applied Effect of carpronium No effect Not applied No effect Noeffect Not applied chloride Effect of cepharanthin Not applied Notapplied Not applied Not applied Not applied Dosage form CNP ointment CNPointment CNP ointment CNP ointment CNP gel Dose 100 μg/g 50 μg/g 100μg/g 100 μg/g 50 μg/g Number of days used 28 days 28 days 2 weeks 6weeks 2 weeks Degree of improvement S1→S0 S1→S1 S3→S2 S2→S0 S1→S0 insymptoms Bald area remained at 1 position at hairline Non-recurrenceperiod 12 months Treatment Treatment 9 months 2 months suspendedcontinuingCase C12

The test subject was a 63 year old female and was a patient with severealopecia areata multilocularis having a hair loss range of S2. There wasa history of allergic rhinitis, and there was coexisting atopicdermatitis. The results of the scratch test were house dust 2+, mite 1+,Dactylis 2+, and ragweed 1+. The alopecia areata of this test subjecthad continued for over one and a half years. This test subject hadreceived application of a steroid and carpronium chloride to the baldarea continuously for 10 months, but there had been no effect, and nohair growth had been observed. This test subject was the same testsubject as the test subject of case B1.

When 100 μg/g BNP gel was applied to the entire bald area of the righttemporal region of this test subject twice a day morning and evening,clear hair growth was observed in 2 weeks. There was no recurrence ofhair loss on the application site even when 8 months had elapsed afterthe application of BNP gel was stopped after 24 days.

However, new multiple bald areas appeared on the left temporal region;100 μg/g CNP ointment was applied twice a day morning and evening,growth of vellus hair was observed after 1 week of application, andclear growth of terminal hair was observed 3 weeks after the applicationwas started. 50 μg/g CNP ointment was applied twice a day morning andevening for 20 days, the hair thickened so as to cover the entire hairloss area, and there was a cure without subsequent application (Ref.FIG. 25). There was no recurrence even when 1 month had elapsed.

Case C13

The test subject was a 38 year old male and was a patient with alopeciaareata multilocularis having a hair loss range of S1. A bald patch hadappeared on the temporal region 2 months earlier, and following this abald area had been observed on the back of the head. There was noprevious history or family history of immune disease. The results of thescratch test of this test subject were house dust 2+, mite 2+, cedar 2+,Dactylis 2+, and ragweed 1+. This test subject had received orallyadministered cepharanthin, but there had been no therapeutic effect.

When 100 μg/g CNP ointment was applied to the entire alopecia of thetemporal region and the back of the head of this test subject twice aday, growth of white hair was confirmed on the entire bald patch after 1week's application, and black hair growth was observed on the peripheralarea (Ref. FIG. 26). This test subject was in a state in which mainlywhite hair had grown and thickened. With regard to this test subject,the hair continued to thicken even when application of the CNP ointmentwas stopped after 1 week, and there was a spontaneous cure. With regardto this test subject, there was no recurrence on the temporal region orthe back of the head even when 1 year had elapsed after application ofthe CNP ointment was stopped.

However, a circular bald area appeared, this time on the crown, 10months after application of the CNP ointment was stopped, and when 50μg/g BNP gel was applied twice a day for 2 weeks, growth of blackterminal hair was observed. However, extensive hair growth was notobserved even when 50 μg/g BNP gel was applied for 3 weeks; when a 50μg/g BCNP:600 μg/mL betamethasone:500 μg/mL gentamicin combination wasapplied once a day for 2 weeks, hair growth was observed earlier thanhad been the case with use of a single agent, the rate of hairlengthening increased, and terminal hair grew extensively.

Case C14

The test subject was a 35 year old female and was a patient withalopecia areata having a hair loss range of S1. This test subject had ahistory of allergic rhinitis, the mother of this test subject was alsoaffected by allergic rhinitis, and this test subject was a case with anatopic predisposition. The results of the scratch test of this testsubject were house dust 2+, mite 2+, cedar 2+, Dactylis 2+, and ragweed1+. A bald patch had appeared on the back of the head of this testsubject 3 months earlier, it subsequently showed a tendency to enlarge,and the entire back of the head had become thin, accompanied by itching.In the case of this test subject, application of a steroid drug hadcaused itchiness, and the hair loss range had enlarged somewhat.

When 100 μg/g CNP ointment was applied to the entire bald patch of theback of the head of this test subject twice a day, growth of white hairand vellus hair was observed after 1 week of application, and growth ofblack terminal hair was clearly observed after 3 weeks. However,although growth of vellus hair was observed in part of the bald area atthe hair growth line of the left back of the head, it did not recover soas to cover the entire bald patch.

Case C15

The test subject was a 52 year old female and was a patient with severealopecia areata having a hair loss range of S3. There was a previoushistory of allergic rhinitis. The results of the scratch test were housedust 1+, cedar 2+, Dactylis 1+, and ragweed 1+. A bald patch hadappeared on the frontal region of this test subject about 1 year earlierwhen doing continuous night shifts, following this there had been atendency for enlargement, and multiple bald areas had appeared over theentire scalp. Application of carpronium chloride to the bald patch hadbeen continued for 8 months, but the hair loss range had only enlargedand there had been no therapeutic effect. This was a case in which therewere no inflammatory symptoms such as erythema, crust, or scale on thescalp of the hair loss area of the test subject. This test subject wasthe same test subject as the test subject of case A3.

When 100 μg/g CNP ointment was applied to the entire bald area of theright temporal region of this test subject twice a day, hair growth wasconfirmed in 1 week, further marked hair growth was observed in 2 weeks,and enlargement of the bald area was completely stopped. Application ofthe CNP ointment was stopped after 14 days, from the next day after that100 μg/g ANP gel was applied twice a day with the expectation of furthereffects, and the therapeutic effect of the CNP ointment continued as itwas; after 5 weeks had elapsed, although application of the ANP gel wascontinuing, thickening of terminal hair and enlargement of the hairgrowth area were seen. However, since this test subject lived far awayfrom the clinic of the present inventor, she could not visit frequently,and the treatment was suspended. Because of this, the bald area of thistest subject remained, and is currently not cured.

Case C36

The test subject was a 57 year old male and was a patient with a seriouscase of alopecia areata having a hair loss range of S2 with coexistingeyebrow hair loss. This test subject had a previous history of atopicdermatitis. The results of the scratch test were house dust 1+, mite 3+,cedar 2+, Dactylis 1+, and ragweed 1+. With regard to this test subject,multiple bald patches had developed about 5 years earlier due to stressat work. This test subject had received liquid nitrogen therapy, but thehair loss range had only spread, and there had been no therapeuticeffect at all. Subsequently, this test subject had been subjected tooral administration and external application of steroid and externalapplication of carpronium chloride for 3 years, but there had not beenany effect.

When 100 μg/g CNP ointment was applied twice a day to the entire baldarea of the right temporal region of this test subject, hair growthmainly of white hair was observed after 3 weeks. Although application ofthe CNP ointment was stopped after 6 weeks, the therapeutic effect ofthe CNP ointment continued thereafter, the hair continued to grow, andthere was a cure. 9 months later, there was no recurrence in this testsubject.

Case C23

The test subject was a 26 year old female and was a patient withalopecia areata monolocularis having a hair loss range of S1. This testsubject had no previous history of alopecia, and none of the family ofthis test subject was a patient with alopecia areata. This test subjecthad developed alopecia areata monolocularis on the back of the head, hadvisited another clinic, and had received application of Rinderon(trademark)-V ointment (Shionogi & Co., Ltd.), which contains 1.2 mg/gbetamethasone valerate, for 6 weeks; there had been a little hairgrowth, but the size of the bald area had increased.

Application of the betamethasone valerate ointment was stopped, andafter being suspended for 4 weeks, when 50 μg/g CNP gel was appliedtwice a day for 2 weeks to a circular bald area of the back of the headof this test subject, marked growth of black terminal hair was observed,and vellus hair became thick and long (FIG. 39).

7. Therapeutic Effect of BNP:Betamethasone:Gentamicin Combination onAlopecia Areata

The therapeutic effects of a BNP:betamethasone:gentamicin combination onalopecia areata are shown in Table 7-2 (case B14).

TABLE 7-2 Case B14 Figure FIG. 36 Gender Female Age 31 years old Whendeveloped 31 years old Hair loss range S1 Treatment site Crown Hair losssite other None than head Family history of None alopecia Family historyof None immune disease Previous history Affected by alopecia areatamultilocularis 10 years earlier. Scratch Test House dust: — Mite: 1+Cedar: 1+ Dactylis: 1+ Ragweed: 2+ Effect of minoxidil Not appliedEffect of steroid drug Not applied Effect of cooling therapy Not appliedAntiallergic drug Not applied Effect of carpronium Not applied chlorideEffect of cepharanthin Not applied Dosage formBNP:betamethasone:gentamicin combination Dose BNP: 50 μg/gbetamethasone: 600 μg/ml gentamicin: 500 μg/ml Number of days used 7days Degree of improvement S1→S0 in symptoms Non-recurrence period 6weeksCase B14

The test subject was a 31 year old female and was a patient withalopecia areata monolocularis having a hair loss range of S1. This testsubject had no family members with alopecia areata. The results of thescratch test of this test subject were house dust −, mite 1+, cedar 1+,Dactylis 1+, and ragweed 2+. She had been affected by alopecia areatamultilocularis 10 years earlier. This time, alopecia areatamonolocularis developed on the crown when work became busy.

When 50 μg/g BNP:600 μg/mL betamethasone:500 μg/mL gentamicincombination was applied to a bald area of the crown of this test subjectfor 1 week, marked hair growth was confirmed (FIG. 36).

8. Therapeutic Effect of CNP:Betamethasone:Gentamicin Combination onAlopecia Areata

The therapeutic effects of a CNP:betamethasone:gentamicin combination onalopecia areata are shown in Table 7-3 (cases C21 and C22).

TABLE 7-3 Case C21 C22 Figure FIG. 37 FIG. 38 Gender Male Female Age 51years old 33 years old When developed 51 years old 33 years old Hairloss range S1 S1 Treatment site Crown Crown Hair loss site other NoneNone than head Family history of None None alopecia Family history ofNone None immune disease Previous history None Affected by alopeciaareata when at primary school and junior high school. Scratch Test Nottested House dust: 2+ Mite: 1+ Cedar: 3+ Dactylis: 2+ Ragweed: 2+ Effectof minoxidil Not applied Not applied Effect of steroid drug No effect Noeffect Effect of cooling therapy Not applied Not applied Antiallergicdrug Not applied Not applied Effect of carpronium No effect No effectchloride Effect of cepharanthin Not applied Not applied Dosage formCNP:betamethasone: CNP:betamethasone: gentamicin gentamicin combinationcombination Dose CNP: 50 μg/g CNP: 50 μg/g betamethasone: betamethasone:600 μg/ml 600 μg/ml gentamicin: gentamicin: 500 μg/ml 500 μg/ml Numberof days used 21 days 14 days Degree of improvement S1→S0 S1→S0 insymptoms Non-recurrence period 2 weeks 7 weeksCase C21

The test subject was a 51 year old male and was a patient with alopeciaareata monolocularis having a hair loss range of S1. This test subjecthad not been affected by alopecia areata in the past, and none of thefamily of this test subject was a patient with alopecia areata. Thistest subject had developed a single circular bald area on the rightcrown about 2 months before visiting the clinic.

‘Dermosol-G Lotion’ (Iwaki Seiyaku Co., Ltd.), which contains 1200 μg/mLbetamethasone valerate and 1000 μg/mL gentamicin sulfate and ‘Calpraninsolution 5%’ (Taiyo Pharmaceutical Industry Co., Ltd.), which contains50 mg/mL carpronium chloride, were applied to the circular bald area ofthe right crown of this test subject twice a day for 2 months, but onlyvellus hair grew, and there was no further hair growth.

The application of Dermosol-G lotion and Calpranin solution 5% wasstopped after 2 months, and when from 3 weeks after that a 50 μg/gCNP:600 μg/mL betamethasone:500 μg/mL gentamicin combination was appliedtwice a day to the circular bald area of the right crown of this testsubject, hair growth was observed after 2 weeks' application, and markedhair growth was observed after 3 weeks' application. Althoughapplication of the CNP:betamethasone:gentamicin combination was stoppedafter 3 weeks, the hair continued to grow even when 1 week had elapsedthereafter, and the skin was hidden (FIG. 37).

Case C22

The test subject was a 33 year old female and visited the clinic when asingle circular bald area appeared on the crown. This test subject was apatient with alopecia areata monolocularis having a hair loss range ofS1. None of the family of this test subject was a patient with alopeciaareata. The results of the scratch test of this test subject were housedust 2+, mite 1+, cedar 3+, Dactylis 2+, and ragweed 2+. This testsubject had been affected by alopecia areata when she was in primaryschool and junior high school.

When 50 μg/g CNP gel was applied twice a day for 2 weeks to the circularbald area of the crown of this test subject, the amount of hair fallingout decreased, the hair became thick, and growth of black terminal hairwas observed.

When application of the CNP gel was stopped after 2 weeks, and from 1week thereafter a 50 μg/g CNP:600 μg/mL betamethasone:500 μg/mLgentamicin combination was applied twice a day for 2 weeks to thecircular bald area of the same site, the amount of hair falling outcontinued to decrease and thick terminal hair continued to grow (FIG.38).

9. Therapeutic Effect of ANP Gel on Androgenetic Alopecia

The therapeutic effects of ANP gel on androgenetic alopecia are shown inTable 8 (cases A8 and A9).

TABLE 8 Case A8 (=B8) A9 (=B11) Figure FIG. 27 Gender Male Male Age 45years old 75 years old When developed From 44 years old About 40 yearsold to 50 years old Treatment site Crown Crown Hair loss state Hairthinning confined to crown, Erythema accompanied by itching erythemaaccompanied by itching and seborrheic scale seen on scalp and seborrheicscale seen on scalp at hair loss site. at hair loss site. Family historyof Father: androgenetic alopecia Father: androgenetic alopeciaandrogenetic alopecia Family history of None None immune diseasePrevious history Atopic dermatitis, allergic Allergic rhinitis rhinitisScratch Test House dust: 2+ House dust: 2+ Mite: 3+ Mite: 2+ Cedar: —Cedar: 3+ Dactylis: 1+ Dactylis: 2+ Ragweed: 1+ Ragweed: 2+ Effect ofminoxidil Not applied No effect Effect of finasteride Not applied Notapplied Effect of glycyrrhetinic Not applied No effect acid Effect ofsteroid drug No effect No effect Effect of carpronium Not applied Notapplied chloride Effect of cepharanthin Not applied Not applied Dosageform ANP gel ANP gel Dose 100 μg/g 100 μg/g Number of days used 2 weeks4 days Degree of improvement Erythema aggravated and became Noimprovement in erythema and in symptoms seborrheic, itching occurred,hair seborrheic scale, no improvement loss range enlarged, no in hairthinning. improvement in seborrheic scale. Non-recurrence period Noimprovement No improvementCase A8

The test subject was a 45 year old male and had suffered from thinninghair confined to the crown since about the age of 44. This test subjecthad coexisting seborrheic alopecia with erythema accompanied by itchingand seborrheic scale on the scalp of the hair loss site. It wasclassified as type II vertex on the Hamilton-Norwood scale. The fatherof this test subject had androgenetic alopecia confined to the crown.The scratch test of this test subject was house dust 2+, mite 3+,Dactylis 1+, and ragweed 1+. This test subject was the same test subjectas the test subject of case B8.

When 100 μg/g ANP gel was applied twice a day morning and evening for 2weeks to the entire hair loss site of the crown of this test subject,the erythema was only slightly improved, hair growth was not observed atall, and in addition itching occurred, erythema appeared, and the hairloss range enlarged. The seborrheic scalp was not markedly improved(Ref. FIG. 27).

Case A9

The test subject was a 75 year old male, and hair on the crown hadbecome thin at around 40 to 50 years old. This test subject showederythema accompanied by itching and seborrheic scale on the scalp of ahair loss site, and had androgenetic alopecia with coexisting seborrheicalopecia. This was classified as type VI on the Hamilton-Norwood scale.The father of this test subject had androgenetic alopecia. The resultsof the scratch test of this test subject were house dust 2+, mite 2+,cedar 3+, Dactylis 2+, and ragweed 2+, and there was a history ofallergic rhinitis. Minoxidil had been applied to this test subject forone year, but there had been no effect. This test subject had also beingusing glycyrrhetinic acid for 2 years, but said that there had been noeffect. This test subject was the same test subject as the test subjectof case B11.

100 μg/g ANP gel was applied to the hair loss site of the crown of thistest subject twice a day morning and evening for 4 days, but improvementin the erythema and seborrheic scale was not observed, and no effect inimproving the alopecia was observed.

10. Therapeutic Effect of BNP Gel on Androgenetic Alopecia

The therapeutic effects of BNP gel on androgenetic alopecia are shown inTable 9-1 (cases B7 to B10) and Table 9-2 (cases B11 to B16).

TABLE 9-1 Case B7 B8 (=A8) B9 B10 Figure FIG. 28-1 FIG. 28-2 Gender MaleMale Male Male Age 39 years old 45 years old 42 years old 65 years oldWhen developed From 38 years old From 44 years old From 40 years old 30sto 40s Treatment site Crown Crown Crown Right frontal region (right M-shaped part) Hair loss state Tingling of crown, Hair thinning Hairthinning Hairline in erythema and scale confined to crown, confined tocrown, frontal region and appeared, hair erythema slight degree offorehead corners thinning on crown. accompanied by erythema and scaleretreated and itching and seen on scalp at there was hair seborrheicscale hair loss site. thinning, wide seen on scalp at range of crown washair loss site. in hair thinning state. Family history of PaternalFather: Father, maternal Father: androgenetic alopecia grandfather:androgenetic grandfather: androgenetic androgenetic alopeciaandrogenetic alopecia alopecia alopecia Family history of Mother: atopicNone None None immune disease dermatitis Previous history Atopicdermatitis Atopic dermatitis, Atopic dermatitis Atopic dermatitisallergic rhinitis Scratch Test House dust: 3+ House dust: 2 + Not testedHouse dust: 2+ Mite: 3+ Mite: 3+ Mite: 2+ Cedar: 1+ Cedar: — Cedar: 1+Dactylis: 2+ Dactylis: 1+ Dactylis: 1+ Ragweed: 1+ Ragweed: 1+ Ragweed:1+ Effect of minoxidil Not applied Not applied No effect Not appliedEffect of finasteride Not applied Not applied Not applied Not appliedEffect of glycyrrhetinic Not applied Not applied Not applied Not appliedacid Effect of steroid drug Not applied? No effect Not applied Notapplied Effect of carpronium Not applied Not applied Not applied Notapplied chloride Effect of cepharanthin Not applied Not applied Notapplied Not applied Dosage form BNP gel BNP gel BNP gel BNP gel Dose 100μg/g 50 μg/g 50 μg/g Started with 50 μg/g, increased to 100 μg/g Numberof days used 10 days 3 weeks 3 weeks 50 μg/g for 3 weeks, then 100 μg/gfor 3 weeks Degree of improvement Terminal hair Erythema, Hair fallingout Only for right- in symptoms grew, hair was seborrheic scale,decreased, vellus hand side to which restored, and hair and itching ofhair hair became dark, BNP gel had been extension loss site markedly andgrowth of applied, vellus promoted. improved, hair terminal hair hairbecame Dandruff falling out observed. terminal hair and disappeared,hair decreased. grew in length thinning range Terminal hair grew,compared with decreased, almost hair was restored, before normal stateand hair thinning application. recovered. clearly improved.Non-recurrence period 8 months Treatment Treatment suspended Treatmentcontinuing continuingCase B7

The test subject was a 39 year old male; the crown had become irritatedat the age of 38, erythema and scale had appeared, the hair of the crownhad become thin, and androgenetic alopecia had developed. This wasclassified as type II vertex on the Hamilton-Norwood scale. The paternalgrandfather of this test subject had androgenetic alopecia. The resultsof the scratch test of this test subject were house dust 3+, mite 3+,cedar 1+, Dactylis 2+, and ragweed 1+.

When 100 μg/g BNP gel was applied to the hair loss site of the crown ofthis test subject twice a day morning and evening, the irritationstarted to disappear after about 1 week, the amount of hair falling outdecreased, after 10 days' application the terminal hair became dark, thearea of thinning hair decreased, and there was recovery to asubstantially normal state. There was no recurrence even when months hadelapsed after application of the BNP gel was stopped after 10 days (Ref.FIG. 28-1 and FIG. 28-2).

Case B8

The test subject was a 45 year old male and had suffered from thinninghair confined to the crown from about 44 years old. This test subjectshowed erythema accompanied by itching and seborrheic scale on the scalpat the hair loss site, and had androgenetic alopecia with coexistingseborrheic alopecia. This was classified as type II vertex on theHamilton-Norwood scale. The father of this test subject had androgeneticalopecia confined to the crown. The results of the scratch test of thistest subject were house dust 2+, mite 3+, Dactylis 1+, and ragweed 1+.This test subject was the same test subject as the test subject of caseA8.

When 100 μg/g ANP gel was applied to the entire hair loss site of thecrown of this test subject twice a day morning and evening for 2 weeks,although the erythema seemed to be slightly improved, the amount of hairfalling out remained large, the seborrheic scalp was not improved, andthe hair loss range enlarged somewhat. Furthermore, itching occurredabout 1 hour after application of the ANP gel, and strong itchingappeared on the 2^(nd) day.

When 50 μg/g BNP gel was applied twice a day morning and evening to theentire hair loss site of the crown of this test subject, erythema,seborrheic scale, and itching on the hair loss site were markedlyimproved on the 1^(st) week after application of the BNP gel wasstarted, the amount of hair falling out decreased to an unnoticeablelevel, the hair clearly thickened objectively, and the thinning hair wasimproved.

When external application of the BNP gel was stopped, with regard tothis test subject, the effect continued for 2 months after the externalapplication was stopped, and a good state in which the amount of hairfalling out was small and there was no itching continued. Furthermore,with regard to this test subject, about 3 months after the externalapplication of the BNP gel was stopped, the volume of hair on the crowndecreased, the thinning hair became conspicuous, the amount of hairfalling out increased, and itchiness occurred.

When, this time, 50 μg/g CNP gel was applied twice a day, this testsubject noticed that blood circulation improved, the amount of hairfalling out started to decrease on about the 5^(th) day, and the itchingdisappeared. With regard to this test subject, the hair started tothicken in about the 3^(rd) week after application of the CNP gel wasstarted, and the hair thickened to a substantially normal state after 7weeks of application. However, for this test subject, if application ofthe CNP gel was stopped for 2 to 3 months, the amount of hair fallingout increased and thinning hair occurred on the crown; the treatment isbeing continued at the present time.

Case B9

The test subject was a 42 year old male and had suffered from thinninghair confined to the crown from about 40 years old. This test subjecthad a slight degree of erythema and scale on the scalp of the hair losssite. This was classified as type V on the Hamilton-Norwood scale. Thefather and the maternal grandfather of this test subject hadandrogenetic alopecia. This test subject was not subjected to a scratchtest.

When 50 μg/g BNP gel was applied twice a day morning and evening to theentire thinning hair site of the crown of this test subject, by the1^(st) week after application was started, the amount of hair fallingout decreased, vellus hair darkened, and growth of terminal hair wasobserved, compared with the state before application in which there wasonly vellus hair. The hair became darker after 3 weeks, and hairthickening could be confirmed objectively.

Case B10

The test subject was a 65 year old male and the hair had thinned whenthe hair line of the frontal region and the corners of the foreheadretreated during his 30s to 40s. The father also had androgeneticalopecia. This was classified as type VI on the Hamilton-Norwood scale.The results of the scratch test of this test subject were house dust 2+,mite 2+, cedar 1+, Dactylis 1+, and ragweed 1+, and there was a historyof atopic dermatitis and allergic conjunctivitis.

Since the thinning hair had left-and-right symmetry, in order toevaluate the effects, 50 μg/g BNP gel was applied twice a day morningand evening to the right front temporal region, that is, the so-calledM-shaped part, and only a gel base was applied to the left-hand side. Bythe 2^(nd) week thickening of vellus hair was observed only on theright-hand side, to which the BNP had been applied, compared with thatbefore application; the concentration of the BNP gel was changed to 100μg/g, it was applied to the right front temporal region for 2 weeks, andvellus hair in the M-shaped part clearly became terminal hair and thickand long compared with that when 50 μg/g BNP gel was applied.

Case B11

The test subject was a 75 year old male and hair on the crown hadthinned from around 40 years old to 50 years old. This test subject hadandrogenetic alopecia with coexisting seborrheic alopecia, in whichthere was erythema accompanied by itching and seborrheic scale on thescalp of the hair loss site. This was classified as type VI on theHamilton-Norwood scale. The father of this test subject had androgeneticalopecia. The results of the scratch test of this test subject werehouse dust 2+, mite 3+, Dactylis 1+, and ragweed 1+. There had been noeffect on this test subject when minoxidil had been applied for 1 year.This test subject had been also using glycyrrhetinic acid for 2 years,but said that there had been no effect. This test subject was the sametest subject as the test subject of case A9.

When 100 μg/g ANP gel was applied to the hair loss site of the crown ofthis test subject twice a day morning and evening for 4 days, there wasno improvement in the erythema or the seborrheic scale.

When 50 μg/g BNP gel was applied to the hair loss site of the crown ofthis test subject twice a day morning and evening, on the 3^(rd) dayafter application was started erythema, seborrheic scale, and itching onthe hair loss site were markedly improved and the amount of hair fallingout become unnoticeable. The hair quality was improved after 2 weeks,the hair became black and thick and darkened, and there was hairthickening. When the BNP gel was also applied to the M-shaped part ofthis test subject twice a day for 20 days, terminal hair grew, and itwas confirmed that growth of hair in the M-shaped part was alsopromoted. This test subject is still being treated with the BNP gel atpresent.

Case B12

The test subject was a 62 year old male who had developed thinning hairon the crown at around 50 years old, and had pityriatic scale anderythema accompanied by itching on the scalp of the hair loss site. Thistest subject had androgenetic alopecia with coexisting alopeciapityroides. This was classified as type III vertex on theHamilton-Norwood scale. The father and a younger brother of this testsubject had androgenetic alopecia. The results of the scratch test ofthis test subject were house dust 2+, mite 3+, cedar 2+, Dactylis 1+,and ragweed 2+, and there was atopic dermatitis and bronchial asthma.The brother had a history of atopic dermatitis and a child had a historyof allergic rhinitis.

When 50 μg/g BNP gel was applied to the entire hair loss site of thecrown of this test subject twice a day morning and evening for 3 weeks,the amount of hair falling out decreased markedly on the 3^(rd) dayafter application of the BNP gel started, and the erythema andseborrheic scale were improved. In the 3^(rd) week the hair became dark,there was hair thickening, and the thinning hair was improvedobjectively (Ref. FIG. 29).

When application of the BNP gel was stopped after 3 weeks, and from thefollowing day 50 μg/g ANP gel was applied twice a day, this test subjectexhibited seborrheic scale, erythema, and itching, the application wasstopped after 1 week.

When from the following day 50 μg/g BNP gel was applied again twice aday, it was as if the seborrheic scale, erythema, and itching due to theANP gel had never happened, the amount of hair falling out decreased,the itching and the seborrheic scale were also improved, the hair becamethick, dark, and long, and there was hair thickening. Since this testsubject had coexisting atopic dermatitis, the body was itchy, but therewas no itchiness on the scalp. With regard to this test subject, evenwhen application of the BNP gel was stopped after 2 weeks, the amount ofhair falling out remained low for 2 months, and only 1 or 2 hairs becameattached to a towel when washing the hair. However, when 2 months hadelapsed after application of the BNP gel was stopped after 2 weeks, theamount of hair falling out started to increase again; when this time 50μg/g CNP gel was applied twice a day, the amount of hair falling outdecreased markedly, the erythema and seborrheic scale were alsoimproved, and the itchiness disappeared. By the 3^(rd) week afterapplication of the CNP gel was started, the hair had become thick, dark,and long, and there was hair thickening.

TABLE 9-2 Case B11 (=A9) B12 B15 B16 Figure FIG. 29 FIG. 40 FIG. 41Gender Male Male Male Male Age 75 years old 62 years old 44 years old 46years old When developed 40s 50s Late 30s Late 30s Treatment site Crown,frontal Crown Frontal region Crown, frontal region region (M-shapedpart) Hair loss Hair on crown Hair on crown Hairline retreated in Hairin frontal state thinned, and thinned, and M shape. region thinned, anderythema accompanied erythema accompanied hair on crown thinned byitching and by itching and after 40 years old. seborrheic scalepityriatic seen on scalp at desquamation seen on hair loss site. scalpat hair loss site. Family history of Father: androgenetic Father,younger Father had Maternal grandfather androgenetic alopecia alopeciabrother: androgenetic alopecia and father had androgenetic androgeneticalopecia alopecia Family history of None Younger brother: None Noneimmune disease atopic dermatitis Child: allergic rhinitis Previoushistory Allergic rhinitis Atopic dermatitis, None None bronchial asthmaScratch Test House dust: 2+ House dust: 2+ Not tested Not tested Mite:2+ Mite: 3+ Cedar: 3+ Cedar: 2+ Dactylis: 2+ Dactylis: 1+ Ragweed: 2+Ragweed: 2+ Effect of minoxidil No effect Not applied Not applied Notapplied Effect of finasteride Not applied Not applied Not applied Notapplied Effect of glycyrrhetinic No effect Not applied Not applied Notapplied acid Effect of steroid drug No effect No effect Not applied Notapplied Effect of carpronium Not applied Not applied Not applied Notapplied chloride Effect of cepharanthin Not applied Not applied Notapplied Not applied Dosage form BNP gel BNP gel BNP gel BNP gel Dose 50μg/g 50 μg/g 100 μg/g 100 μg/g 200 μg/g Number of days used 3 weeks 20days 3 weeks 3 weeks + 2 weeks Degree of improvement Erythema,seborrheic Erythema, pityriatic Hair falling out Hair loss area of insymptoms scale, and itching desquamation, and decreased, and hair crowndecreased. of hair loss site itching of hair loss thickened. Hairs Hairsbecame thick, markedly improved, site markedly became thick and terminalhair grew hair falling out improved, hair long, and black and restored,and decreased, and hair falling out terminal hair grew in feeling ofvolume quality improved. decreased markedly, hair thinning part ofincreased. Hair in Hairs became thick, and hair became dark M-shapedpart. frontal region became black, and dense. and thickened. thick andhair Terminal hair grew thickening observed. in M-shaped part and becamedark. Non-recurrence period Treatment continuing Treatment continuingTreatment continuing Treatment continuingCase B15

The test subject was a 44 year old male who had developed M-shapedandrogenetic alopecia in his late 30s. This was classified as type IIIon the Hamilton-Norwood scale and this was a so-called M-shaped hairloss case. The father of this test subject had androgenetic alopecia.

When 100 μg/g BNP gel was applied to the frontal region of this testsubject twice a day for 2 weeks, growth of black terminal hair wasobserved on the M-shaped thinning hair part after 5 days, and it becamemore marked in the 2^(nd) week (FIG. 40).

When application of the BNP gel was stopped after 2 weeks, and from the5^(th) day after that a 50 μg/g BNP:600 μg/mL betamethasone:500 μg/mLgentamicin combination was applied twice a day for 1 week, the amount ofhair falling out further decreased, the number of hairs lost duringwashing decreased to 1 to 2 hairs, and the hair became thicker. Due tothis test subject being busy, the application was stopped, and even when3 weeks had elapsed, the thickened hair state was certainly maintainedcompared with that before application.

Case B16

The test subject was a 46 year old male; thinning hair had occurred inthe frontal region from the late 30s, and thinning hair occurred also onthe crown after the age of 40. This test subject was classified as typeV on the Hamilton-Norwood scale, and both the father and the maternalgrandfather had androgenetic alopecia.

When 100 μg/g BNP gel was applied to the frontal region and the crown ofthis test subject twice a day for 3 weeks, marked growth of blackterminal hair was observed on the crown, the feeling of volume of thehair increased, and the hair loss range on the crown decreased. The hairloss range of the crown at this point was 4 cm×3.4 cm (FIG. 41).

When the application of 100 μg/g BNP gel was stopped after 3 weeks andfrom the following day 200 μg/g BNP gel was applied twice a day for 2weeks, the hair further thickened and restored, and the hair loss rangedecreased to 2.5 cm×2.4 cm. Furthermore, hair on the frontal region, forwhich clear improvement could not be observed by application of 100 μg/gBNP gel, became thick and dark with 2 weeks' application of 200 μg/g BNPgel, and the number of terminal hairs increased (FIG. 41).

11. Therapeutic Effect of CNP Ointment on Androgenetic Alopecia

The therapeutic effects of CNP ointment on androgenetic alopecia areshown in Table 10 (cases C16 and C17).

TABLE 10-1 Case C16 C17 Figure FIG. 30-1 FIG. 31 FIG. 30-2 Gender MaleMale Age 56 years old 59 years old When developed Late 30s 50s Treatmentsite Crown, frontal region Crown, frontal region Hair loss state Hairthinning with vellus hair Hair thinning from crown to seen from crown tofrontal frontal region. Scalp at hair region. loss site had erythema,scale, abrasion scars, and strong itching sensation. Family history ofFather, paternal grandfather: Father, grandfather: androgeneticandrogenetic alopecia androgenetic alopecia alopecia Family history ofNone None immune disease Previous history Atopic dermatitis DiabetesScratch Test House dust: — House dust: 2+ Mite: 1+ Mite: 3+ Cedar: —Cedar: — Dactylis: — Dactylis: 2+ Ragweed: — Ragweed: 2+ Effect ofminoxidil No effect Not applied Effect of finasteride Not applied Notapplied Effect of glycyrrhetinic Not applied Not applied acid Effect ofsteroid drug Not applied No effect Effect of carpronium Not applied Notapplied chloride Effect of cepharanthin Not applied Not applied Dosageform CNP ointment CNP ointment Dose 100 μg/g 50 μg/g Number of days used28 days 14 days Degree of improvement Hair falling out decreased,Itching subsided, erythema and in symptoms vellus hair turned intoterminal abrasion scars on scalp hair, thick and dark. disappeared, andshort hair grew in frontal region and crown. Non-recurrence periodTreatment continuing Treatment continuingCase C16

The test subject was a 56 year old male with androgenetic alopecia inwhich the hair from the crown to the frontal region had become thin andvellus since his 40s. This test subject was classified as type Va on theHamilton-Norwood scale and was a case in which there was both theso-called M-shaped and O-shaped hair loss sites. 1% minoxidil hadpreviously been applied to this test subject for many years, but therehad been no effect, and because recently there had been redness withitchiness, its use had been stopped. In the case of this test subject,the hair had become thin without inflammatory symptoms such as erythemaor scale on the scalp of the hair loss site. The father and the paternalgrandfather of this test subject had androgenetic alopecia. The resultsof the scratch test of this test subject were house dust −, mite 1+,cedar −, Dactylis −, and ragweed −.

When 100 μg/g CNP ointment was applied to the entire hair loss site ofthe crown and the frontal region of this test subject twice a daymorning and evening, the amount of hair falling out decreased after 2weeks of application, apparent improvement effects were observed, vellushair became dark, and vellus hair clearly grew and became dark after 4weeks of application (Ref. FIGS. 30-1 and 30-2). With regard to thistest subject, when treatment with the CNP ointment was stopped after ithad continued for 8 weeks, the therapeutic effect on hair loss wasgradually lost after 3 months has elapsed subsequent to stoppingapplication.

When 50 μg/g BNP gel was applied to the entire hair loss site of thecrown and the frontal region of this test subject once a day for 1month, fine hair became dark and thick, and the amount of hair fallingout decreased.

After 2 months had elapsed since the application of 50 μg/g BNP gel wasstarted, it was changed to application of 200 μg/g BNP gel once a day,and when the application had continued for 1 week, hair started to riseand the rate of hair lengthening increased compared with the case with50 μg/g BNP gel. The hair of this test subject had become white 5 yearsearlier if it was not dyed, but black hair thickened and lengthened inalmost all of the frontal region, which was an area to which the BNP gelhad been applied.

Case C17

The test subject was a 59 year old male with androgenetic alopecia inwhich the hair had become thin from the crown to the frontal region.This test subject was classified as type VII on the Hamilton-Norwoodscale, and was a case in which there were both M-shaped and O-shapedhair loss sites. In the case of this test subject, there were erythema,scale, and abrasion scars on the scalp at the hair loss site, and theitching sensation was strong. The father and a grandfather of this testsubject had androgenetic alopecia. The results of the scratch test ofthis test subject were house dust 2+, mite 3+, cedar −, Dactylis 2+, andragweed 2+. This test subject had previously been prescribed a steroidand had continued with its external use for 7 months, but the itchingsensation recurred after only about 1 hour, the inflammation did notcalm down, and there was no improvement.

When 50 μg/g CNP ointment was applied to the entire hair loss site ofthis test subject twice a day for 1 week, the itching calmed down,erythema and abrasion scars on the scalp disappeared, and accompanyingthis there was growth of short hair on the crown and the frontal region(Ref. FIG. 31). This test subject is currently still being treated withthe CNP ointment, and the application of CNP ointment had continued for14 days at the time of filing of this patent application.

12. Therapeutic Effect of CNP Gel on Androgenetic Alopecia

The therapeutic effects of CNP gel on androgenetic alopecia are shown inTable 10-2 (cases C24 and C37 to C39).

TABLE 10-2 Case C24 (=C40) C37 C38 C39 Figure FIG. 42 Gender Male MaleMale Male Age 48 years old 43 years old 33 years old 34 years old Whendeveloped 5 year earlier 40s 30s Late 20's Treatment site Crown Frontalregion, Frontal region Frontal region crown Hair loss state Hairline infrontal Thinning hair in Thinning hair on Thinning hair on regionretreated in front temporal crown, and hair crown, and hair M shape andbecame region and crown. loss in front loss in front vellus hair, andtemporal region. temporal region. thinning hair on crown. Family historyof None None Father and maternal Father: androgenetic alopeciagrandfather: androgenetic androgenetic alopecia alopecia Family historyof Older brother: Unknown Unknown Unknown immune disease atopicdermatitis and allergic rhinitis Previous history Allergic rhinitisUnknown Unknown Unknown and allergic conjunctivitis Scratch Test Housedust: 2+ Not tested Not tested Not tested Mite: 3+ Cedar: 2+ Dactylis:2+ Ragweed: 2+ Effect of minoxidil Not applied Not applied Not appliedNot applied Effect of finasteride Not applied Not applied Not appliedNot applied Effect of glycyrrhetinic Not applied Not applied Not appliedNot applied acid Effect of steroid drug Not applied Not applied Notapplied Not applied Effect of carpronium Not applied Not applied Notapplied Not applied chloride Effect of cepharanthin Not applied Notapplied Not applied Not applied Dosage form CNP gel CNP gel CNP gel CNPgel Dose 50 μg/g 100 μg/g 50 μg/g 50 μg/g Number of days used 1 week 2weeks 2 weeks 3 weeks Degree of improvement Hair falling out Hairfalling out Hair thickening Hair thickening in symptoms decreased, hairdecreased, hair seen on crown, and seen on crown, and became thick, andquality became vellus hair became hair became thick the number of hairsthick, and hair thick and dark. In and dark. In M- increased. thinningon crown M-shaped hair shaped part, hair improved. thinning part, grewand hair was vellus hair became restored at slow thick and long, andpace. hair falling out decreased. Non-recurrence period TreatmentTreatment Treatment Treatment continuing continuing continuingcontinuingCase C24

The test subject was a 48 year old male and had androgenetic alopecia.With regard to this test subject, 5 years earlier, the hair line in thefrontal region had retreated in an M-shape and had become vellus hair,and the hair on the crown had become thin. This was classified as typeIII vertex on the Hamilton-Norwood scale. None of the family of thistest subject had thinning hair.

When 50 μg/g CNP gel was applied twice a day to the crown of this testsubject for 1 week, the amount of hair falling out decreased, the hairbecame thick, and the number of hairs increased (FIG. 42).

When application of the CNP gel was stopped after 1 week and a 50 μg/gCNP:600 μg/mL betamethasone:500 μg/mL gentamicin combination was thenapplied twice a day for 1 week, the amount of hair falling out furtherdecreased, and the hair became thicker. Furthermore, hair growth wasobserved also in the M-shaped part 2 weeks after application of theCNP:betamethasone:gentamicin combination was started.

After the application was suspended for 3 weeks, when a 5% minoxidilsolution was applied and 100 μg/g CNP gel was then applied as amultilayer application, there were no symptoms of irritation, the hairgradually became thick and there was restoration of terminal hair bothin the M-shaped part and the crown after 1 week following starting themultilayer application.

Case C37

The test subject was a 43 year old male and had androgenetic alopecia inwhich thinning hair had appeared on the front temporal region and thecrown from his 40s. This test subject was classified as type V on theHamilton-Norwood scale, and was a case having both so-called M-shapedand O-shaped hair loss sites.

When 100 μg/g CNP gel was applied to the frontal region and the crown ofthis test subject twice a day for 2 weeks, the amount of hair fallingout decreased, the number of hairs falling became about 10 when washingthe hair, the quality of the hair changed from being fine to beingthick, the hair became long, the thinning hair on the crown wasimproved, and it became difficult to see through to the skin.

Case C38

The test subject was a 33 year old male whose hair quality had becomecurly and fine in his late 20s, and androgenetic alopecia had developedin his 30s. This was classified as type III vertex on theHamilton-Norwood scale, and there was so-called M-shaped alopecia inwhich the hair on the crown became thin and the front temporal regionhad hair loss. The father and the maternal grandfather of this testsubject had androgenetic alopecia.

When 50 μg/g CNP gel was applied to the frontal region of this testsubject twice a day for 1 week, the amount of hair falling outdecreased, and hair did not become attached to a pillow. Hair thickeningon the crown of this test subject was confirmed by the 2^(nd) week, andvellus hair became thick and dark. This test subject said that when heentered a freezer at work, he subjectively did not feel cold airpenetrating the scalp. With regard to this test subject, vellus hair inthe M-shaped thinning hair part also became thick and long. With regardto this test subject, when application was subsequently continued, thenumber of terminal hairs on the crown increased considerably and thehair became longer after 2 months had elapsed, there was hairthickening, and thinning hair became inconspicuous. Even in the M-shapedthinning hair part, although the hair was finer than that on the crown,terminal hair rather than vellus hair increased, became longer, and thenumber of long hairs increased.

Subsequently, when a 50 μg/g CNP:600 μg/mL betamethasone:500 μg/mLgentamicin combination was applied twice a day for 2 weeks, the hairfurther thickened on the crown and the hair on the M-shaped part becamelonger and thicker, but there was accompanying folliculitis, which wasthought to be a side effect of a steroid.

Case C39

The test subject was a 34 year old male whose hair had become thin inhis late 20's and who had androgenetic alopecia. This was classified astype V on the Hamilton-Norwood scale, and there was thinning hair on thecrown and so-called M-shaped alopecia in which there was hair loss onthe front temporal region. The father of this test subject hadandrogenetic alopecia. A 1% minoxidil solution had been applied to thistest subject for 1 year, but there had been no effect. Furthermore,biotin and a Chinese medicine had been orally administered to this testsubject, but there had been no effect.

When 50 μg/g CNP gel was applied to the frontal region of this testsubject twice a day for 3 weeks, hair thickening was observed on thecrown, and the hair became thick and dark. On the M-shaped part alsohair growth and hair restoration were observed at a slow pace.

13. Therapeutic Effect of CNP:Betamethasone:Gentamicin Combination onAndrogenetic Alopecia

The therapeutic effects of CNP:betamethasone:gentamicin combination onandrogenetic alopecia are shown in Table 10-3 (case C25).

TABLE 10-3 Case C25 Figure FIG. 43, FIG. 44 Gender Male Age 75 years oldWhen developed 5 years earlier Treatment site Frontal region and crownHair loss state Hairline in frontal region retreated in M-shape andbecame vellus hair, and thinning hair on crown. From half a year earlierthere had been coexisting alopecia pityroides accompanied by scale,erythema, and itching. Family history of None androgenetic alopeciaFamily history of Unknown immune disease Previous history UnknownScratch Test Unknown Effect of minoxidil Not applied Effect offinasteride Not applied Effect of glycyrrhetinic Not applied acid Effectof steroid drug No effect Effect of carpronium Not applied chlorideEffect of cepharanthin Not applied Dosage formCNP:betamethasone:gentamicin combination Dose 25 μg/g CNP 600 μg/mlbetamethasone 500 μg/ml gentamicin Number of days used 12 days Degree ofimprovement Hair falling out decreased, scale and erythema in symptomsimproved, and marked hair thickening in both crown and M-shaped part.Hair became thick and long. Non-recurrence period UnknownCase C25

The test subject was a 75 year old male who had androgenetic alopecia.With regard to this test subject, the hair line in the frontal regionhad retreated in an M-shape and the crown had started to become thinfrom 5 years earlier. This test subject had scale and erythema withaccompanying itching, and had coexisting alopecia pityroides. This testsubject was a case having both the so-called M-shaped and O-shaped hairloss sites, and was classified as type VI on the Hamilton-Norwood scale.

When a 25 μg/g CNP:600 μg/mL betamethasone:500 μg/mL gentamicincombination was applied to the frontal region and the crown of this testsubject twice a day for 12 days, there was almost no hair falling out,and the scale and erythema disappeared completely. Application of theCNP:betamethasone:gentamicin combination was stopped after 12 days, andwhen observation was made 18 days after application had been stopped,the hair quality had improved for both the hair line of the frontalregion and the crown, the hair had become thick and long, and there wasmarked hair thickening (FIG. 43 and FIG. 44).

14. Therapeutic Effect of CNP:Clobetasol Combination on AndrogeneticAlopecia

The therapeutic effects of CNP:clobetasol combination on androgeneticalopecia are shown in Table 10-4 (case C26).

TABLE 10-4 Case C26 Figure FIG. 45 Gender Male Age 61 years old Whendeveloped Unknown Treatment site Frontal region and crown Hair lossstate Hair thinning was conspicuous in frontal region and crown. Familyhistory of Father, paternal grandfather, and maternal androgeneticalopecia grandfather had androgenetic alopecia. Family history ofUnknown immune disease Previous history Bronchial asthma Scratch Test —Effect of minoxidil Not applied Effect of finasteride Not applied Effectof glycyrrhetinic Not applied acid Effect of steroid drug No effectEffect of carpronium Not applied chloride Effect of cepharanthin Notapplied Dosage form CNP:clobetasol combination Dose 50 μg/g CNP 250 μg/gclobetasol Number of days used 7 days Degree of improvement Markedgrowth of black terminal hair, itching in symptoms sensationdisappeared, and dandruff stopped. Non-recurrence period TreatmentcontinuingCase C26

The test subject was a 61 year old male who had androgenetic alopecia.This test subject was a case in which the frontal region and the crownhad thinning hair mixed with white hair and there was coexistingalopecia pityroides accompanied by itching and scale. The father, thepaternal grandfather, and the maternal grandfather of this test subjecthad androgenetic alopecia. This test subject was affected by bronchialasthma. The test results of the scratch test of this test subject wereas given, and this test subject was a case having both the so-calledM-shaped and O-shaped hair loss sites, and was classified as type IVa onthe Hamilton-Norwood scale. ‘Dermovate Scalp Lotion 0.05%’(GlaxoSmithKline plc), which contains 0.05% of clobetasol propionate and‘Fulmeta Lotion’ (Shionogi & Co., Ltd.), which contains 0.1% ofmometasone furancarboxylate had been applied to the frontal region andthe crown of this test subject for 9 months, but there had been noeffect at all, and the hair had become somewhat thin.

When a 50 μg/mL CNP:250 μg/g clobetasol combination was applied to thefrontal region and the crown of this test subject twice a day for 1week, marked growth of black terminal hair was observed, the itchingsensation disappeared, and dandruff disappeared (FIG. 45).

When the application of 50 μg/mL CNP:250 μg/g clobetasol combination wasstopped after 1 week, and from the following day a 50 μg/mL BNP:250 μg/gclobetasol combination was applied twice a day, growth of black terminalhair became more marked after 1 week, the amount of hair falling outdecreased markedly after 2 weeks, and black terminal hair lengthened ata very high rate.

15. Therapeutic Effect of CNP:Carpronium Chloride Combination onAndrogenetic Alopecia

The therapeutic effects of CNP:carpronium chloride combination onandrogenetic alopecia are shown in Table 10-5 (case C27).

TABLE 10-5 Case C27 Figure FIG. 46 Gender Male Age 32 years old Whendeveloped About 30 years old Treatment site Frontal region, lefttemporal region Hair loss state Hair loss in M-shaped part of frontalregion, and circular bald area in left temporal region. Family historyof None androgenetic alopecia Family history of None immune diseasePrevious history None Scratch Test Not tested Effect of minoxidil Notapplied Effect of finasteride Not applied Effect of glycyrrhetinic Notapplied acid Effect of steroid drug Not applied Effect of carpronium Notapplied chloride Effect of cepharanthin Not applied Dosage formCNP:carpronium chloride combination Dose 50 μg/g Number of days used 14days Degree of improvement Larger number of black terminal hairs grew insymptoms than when single agent was applied. Non-recurrence periodTreatment continuingCase C27

The test subject was a 32 year old male who had androgenetic alopecia.With regard to this test subject, hair of the M-shaped hair line in thefrontal region suddenly thinned at the age of 30 and retreated. Alopeciaareata monolocularis had also developed in the left temporal region 6weeks before visiting the clinic. None of the family of this testsubject had alopecia. This test subject was classified as type III onthe Hamilton-Norwood scale, and had so-called M-shaped androgeneticalopecia.

When 50 μg/g CNP gel was applied twice a day to an androgenetic alopeciaarea of the frontal region and a circular bald area of the left temporalregion of this test subject, growth of black terminal hair was observedafter 2 weeks both in the M-shaped hair line area and the circular baldarea (FIG. 46).

When application of the CNP gel was stopped after 2 weeks and from thefollowing day a CNP:carpronium chloride combination was applied twice aday, a larger amount of black terminal hair than with the single agentgrew after 1 week.

16. Therapeutic Effect of Multilayer Application of CNP Gel andMinoxidil on Androgenetic Alopecia

The therapeutic effects of multilayer application of CNP gel andminoxidil on androgenetic alopecia are shown in Table 10-6 (case C40).

TABLE 10-6 Case C40 (=C24) Figure Gender Male Age 48 years old Whendeveloped 5 years earlier Treatment site Frontal region, crown Hair lossstate Hairline in frontal region retreated in M-shape and became vellushair, thinning hair on crown. Family history of None androgeneticalopecia Family history of Older brother: atopic dermatitis and allergicimmune disease rhinitis Previous history Allergic rhinitis and allergicconjunctivitis Scratch Test House dust: 2+ Mite: 3+ Cedar: 2+ Dactylis:2+ Ragweed: 2+ Effect of minoxidil Not applied Effect of finasteride Notapplied Effect of glycyrrhetinic Not applied acid Effect of steroid drugNot applied Effect of carpronium Not applied chloride Effect ofcepharanthin Not applied Dosage form Multilayer application of CNP geland minoxidil Dose 100 μg/g CNP gel 1% minoxidil Number of days used 1week Degree of improvement Restoration of terminal hair seen in M-shapedin symptoms part and crown. Non-recurrence period Treatment continuingCase C40

Case C40 was the same test subject as C24 and was an androgeneticalopecia case. Details of this case are as described for case C24.

When 5% minoxidil solution was applied to this test subject and afterthat 100 μg/g CNP gel was applied by multilayer application, there wereno symptoms of irritation, and restoration of black terminal hair wasobserved, although it was gradual, on the M-shaped part and the crownfrom 1 week after starting the multilayer application.

17. Therapeutic Effect of Multilayer Application of BNP Gel andMinoxidil on Androgenetic Alopecia

The therapeutic effects of multilayer application of BNP gel andminoxidil on androgenetic alopecia are shown in Table 10-7 (case B29).

TABLE 10-7 Case B29 Figure Gender Male Age 52 years old When developedAbout 23 years old Treatment site Crown Hair loss state Frontal regionand crown had thinning hair. Family history of None androgeneticalopecia Family history of None immune disease Previous history NoneScratch Test Not tested Effect of minoxidil Not applied Effect offinasteride Not applied Effect of glycyrrhetinic Not applied acid Effectof steroid drug Not applied Effect of carpronium Not applied chlorideEffect of cepharanthin Not applied Dosage form Multilayer application ofBNP gel and minoxidil Dose 100 μg/g BNP gel 5% minoxidil Number of daysused 1 week Degree of improvement No symptoms of irritation, hair becamesupple, in symptoms and hair thinning on crown improved. Non-recurrenceperiod Treatment continuingCase B29

The test subject was a 52 year old male and had androgenetic alopeciathat was classified as type IV on the Hamilton-Norwood scale. This testsubject had shown thinning hair in the frontal region at around 23 yearsold, and the crown currently had thinning hair. This test subject had nofamily members with alopecia. Oral administration of Hangekobokuto hadnot shown any effect on this test subject.

When 100 μg/g BNP gel was applied twice a day to the androgeneticalopecia area of the crown of this test subject, the amount of hairfalling out decreased dramatically 2 weeks later, the hair on the crownbecame thick and dark, and growth of black terminal hair was observed.However, after that, when application of the BNP gel was suspended for 3weeks, the hair became less robust and less resilient.

When multilayer application of 5% minoxidil solution and then 100 μg/gBNP gel was carried out for 1 week, there were no symptoms ofirritation, the hair became robust, and the thinning hair on the crownwas improved.

Case C32

Case C32 was a case of coexisting androgenetic alopecia and seborrheicalopecia, and was the same as cases A9, B11, and B22.

When 100 μg/g BNP gel was applied to thinning hair sites of the crownand the M-shaped part of this test subject twice a day morning andevening, erythema, seborrheic scale, and itching of the hair loss sitesmarkedly improved on the 3^(rd) day after application was started, andthe amount of hair falling out become unnoticeable. With regard to thistest subject, the hair quality was improved after 2 weeks, the hairbecame thick and dark, and there was hair thickening. With regard tothis test subject, on the 2^(nd) week of application, in the M-shapedpart also, vellus hair became thick, dark, and although it was slow thehair became long.

In order to examine an external preparation that could further reducethe thinning hair range of the crown of this test subject, after 5%minoxidil solution was applied to the crown and the M-shaped part, 100μg/g BNP gel was applied by multilayer application; the hair on thecrown became black and dark compared with application of a single BNPagent when 1 week had elapsed after the multilayer application hadstarted, and there was hair thickening. This test subject is stillhaving multilayer application treatment with 5% minoxidil and 100 μg/gBNP gel at the present.

18. Therapeutic Effect of ANP Gel on Postpartum Alopecia

The therapeutic effects of ANP gel on postpartum alopecia are shown inTable 10-8 (case A10).

TABLE 10-8 Case A10 Figure FIG. 47 Gender Female Age 45 years old Whendeveloped 44 years old Treatment site Crown Hair loss state Thinninghair on crown was conspicuous. Hair loss site other None than headFamily history of Unknown alopecia Family history of Unknown immunedisease Previous history None Scratch Test Not tested Effect ofminoxidil Not applied Effect of glycyrrhetinic Not applied acid Effectof steroid drug Not applied Effect of cooling therapy Not appliedAntiallergic drug No effect Effect of carpronium Not applied chlorideEffect of cepharanthin Not applied Dosage form ANP gel Dose 100 μg/gNumber of days used 21 days Degree of improvement Hair falling out didnot decrease, but hair loss in symptoms range of crown enlarged.Non-recurrence period Treatment continuingCase A10

This test subject was a 45 year old female and had developed thinninghair on the crown after childbirth.

When 100 μg/g ANP gel was applied to the crown of this test subjecttwice a day for 3 weeks, the amount of hair falling out did notdecrease, but the hair loss range on the crown enlarged somewhat.

When application of the ANP gel was stopped after 3 weeks and suspendedfor 6 months, and then 50 μg/g BNP gel was applied to the hair loss siteof the crown once every 3 to 4 days for 3 weeks, the thinning hair onthe crown thickened to a level such that it was visually unnoticeable(FIG. 47).

19. Therapeutic Effect of BNP Gel on Postpartum Alopecia

The therapeutic effects of BNP gel on postpartum alopecia are shown inTable 10-9 (case B17).

TABLE 10-9 Case B17 Figure FIG. 47 Gender Female Age 45 years old Whendeveloped 44 years old Treatment site Crown Hair loss state Thinninghairon crown was conspicuous. Hair loss site other than head None Familyhistory of alopecia Unknown Family history of immune disease UnknownPrevious history None Scratch Test Not tested Effect of minoxidil Notapplied Effect of glycyrrhetinic acid Not applied Effect of steroid drugNot applied Effect of cooling therapy Not applied Antiallergic drug Noeffect Effect of carpronium chloride Not applied Effect of cepharanthinNot applied Dosage form BNP gel Dose 50 μg/g Number of days used 21 daysDegree of improvement in symptoms Hair grew on crown, thinning hairimproved to a level such that it was unnoticeable Non-recurrence period2 monthsCase B17

Case B17 was the same test subject as case A10.

When, 6 months after application of the ANP gel was stopped, 50 μg/g BNPgel was applied to the crown 3 to 4 times a day for 3 weeks, theterminal hair thickened, the amount of hair falling out decreased, andthe thinning hair recovered to the extent that it was unnoticeable (FIG.47).

20. Therapeutic Effect of CNP Gel on Postpartum Alopecia

The therapeutic effects of CNP gel on postpartum alopecia are shown inTable 11-1 (case C18).

TABLE 11-1 Case C18 Figure FIG. 32 Gender Female Age 27 years old Whendeveloped 27 years old Treatment site Crown Hair loss state Thinninghair from crown to frontal region was conspicuous. Hair loss site otherthan head None Family history of alopecia Mother: androgenetic alopeciain female with thinning on crown Family history of immune diseaseMother: allergic rhinitis Previous history Atopic dermatitis ScratchTest House dust: 3+ Mite: 2+ Cedar: 2+ Dactylis: 2+ Ragweed: 2+ Effectof minoxidil Not applied Effect of steroid drug No effect Effect ofcooling therapy Not applied Antiallergic drug Not applied Effect ofcarpronium chloride No effect Effect of cepharanthin Not applied Dosageform CNP gel Dose 100 μg/g Number of days used 7 days Degree ofimprovement in symptoms Hair parting was wide and conspicuous, but shortsturdy terminal hair grew, and bald area along hair parting improved.Non-recurrence period 6 monthsCase C18

The test subject was a 27 year old female and was a patient withpostpartum alopecia. This was a case with a history of atopic dermatitisand with an atopic predisposition. The mother of this test subject wasaffected by allergic rhinitis, and the mother of this test subject alsohad female pattern alopecia with a thin crown. The results of thescratch test were house dust 3+, mite 3+, cedar 2+, Dactylis 2+, andragweed 2+. This test subject had rapidly lost hair due to childbirth 7months earlier. Thinning hair on the crown and the frontal region wasconspicuous for this test subject. This test subject had receivedapplication of a steroid and carpronium chloride for 5 months, but therewas still a large amount of hair falling out, and thinning hair on thecrown had not improved.

When 100 μg/g CNP gel was applied to the entire crown of this testsubject twice a day, the amount of hair falling out decreaseddramatically after 7 days of application, it improved such that only 5to 6 hairs fell out per day, and the seborrheic scalp was also improved(Ref. FIG. 32). A large amount of short terminal hair grew from thefrontal region to the crown, the thinning hair of the crown becameinconspicuous objectively, and a substantially normal state wasrecovered. However, the amount of hair falling out increased around thesummer when half a year had elapsed since application of the CNP gel wasstopped after 7 days, and the crown showed thinning hair.

When this time 100 μg/g BNP gel was applied twice a day to the entirecrown, the amount of hair falling out decreased dramatically after 7days' application, and a large amount of short terminal hair grew.Application of the BNP gel was stopped after 7 days, but there was norecurrence for half a year thereafter.

21. Therapeutic Effect of BNP Gel on Female Pattern Alopecia

The therapeutic effects of BNP gel on female pattern alopecia are shownin Table 11-2 (cases B18 to B20 and B27).

TABLE 11-2 Case B18 B19 B20 B27 Figue None FIG. 48 FIG. 49 Gender FemaleFemale Female Female Age 46 years old 59 years old 50 years old 66 yearsold When developed 46 years old 59 years old About 47 years old 65 yearsold Treatment site Crown Frontal region Crown Crown, frontal region,left M- shaped part Hair loss state Diffuse hair loss Thinning hair inHair thinning on Thinning hair in on and around the hair parting incrown and its wide range from mid line. frontal region was periphery,large crown to frontal conspicuous. amount of hair region and left M-falling out. shaped part, the skin showed through. Seborrheic scale wasconspicuous. The amount of hair falling out was very large. Hair losssite None None None None other than head Family history of Father, NoneMother had female 65 years old alopecia grandfather, and patternalopecia and older brother had father had androgenetic androgeneticalopecia alopecia Family history of Grandfather, Older None None Noneimmune disease brother: asthma Previous history Atopic dermatitis NoneNone None bronchial asthma allergic rhinitis Scratch Test House dust: 2+Not tested Not tested Not tested Mite: 2+ Cedar: — Dactylis: — Ragweed:— Effect of Not tested Not tested Not tested Not tested minoxidil Effectof steroid Not applied Not applied No effect No effect drug Effect ofcooling Not applied Not applied Not applied Not applied therapyAntiallergic drug Not applied Not applied Not applied No effect Effectof Not applied Not applied Not applied Not applied carpronium chlorideEffect of Not applied Not applied Not applied Not applied cepharanthinDosage form BNP gel BNP gel BNP gel BNP gel Dose 50 μg/g 100 μg/g 50μg/g 50 μg/g Number of days 28 days 14 days 14 days 21 days used Degreeof After BNP gel was Hair thinning in Hair falling out By 10^(th) dayafter improvement in applied for 2 frontal region decreased to oneapplication twice a symptoms weeks, marked hair improved, and slightthird, and hair day for 5 days, growth and degree of erythema thinningbecame black terminal hair restoration was disappeared. inconspicuous.grew on hairline and observed, and hair crown, seborrheic thickened sothat scale on scalp was no skin could be markedly improved, seen. Thenumber and hair falling out of hairs falling decreased. out duringshampooing decreased to about 10. Non-recurrence >4 months 1 month 1month 2 weeks periodCase B18

The test subject was a 56 year old female and was a patient with femalepattern alopecia. This test subject had a large amount of broken hairand hair falling out, and diffuse hair loss was observed centered on themid line. The father, a grandfather, and an older brother of this testsubject had androgenetic alopecia.

When 50 μg/g BNP gel was applied to the crown of this test subject,marked hair growth and hair restoration were observed about 2 weeksafter the application was started, and during application the amount ofhair falling out and sticking to the hands when shampooing decreased toabout 10 hairs. With regard to this test subject, the hair thickened toan extent such that eventually the skin could not be seen. There was nosubsequent external application for 4 months, the terminal hairremained, and a state with a small amount of hair falling out could bemaintained.

Case B19

The test subject was a 59 year old female and was a patient with femalepattern alopecia. This test subject had developed conspicuous thinninghair at the hair parting in the frontal region in her 50s.

When 100 μg/g BNP gel was applied twice a day for 2 weeks, the thinninghair thickened and became inconspicuous to a degree that could be saidto be normal, and the application was therefore stopped.

However, half a year later, thinning hair at the hair parting in thefrontal region became conspicuous again, and when this time 50 μg/g CNPgel was applied twice a day for 1 week, the thinning hair thickened toan extent such that it was unnoticeable (FIG. 48).

When a 50 μg/g CNP:600 μg/mL betamethasone:500 μg/mL gentamicincombination was further applied twice a day, during the 1^(st) week astate in which the amount of hair falling out was unchanged wasmaintained and there was no itching, but during the 2^(nd) week a slightdegree of erythema occurred and it became possible to see through to thescalp slightly. The application of 50 μg/g CNP:600 μg/mLbetamethasone:500 μg/mL gentamicin combination was therefore stoppedafter 10 days, and when from the following day it was changed to 100μg/g CNP gel, the hair thickened again with application twice a day for1 week.

However, when the application of 100 μg/g CNP gel was stopped after 1week and 100 μg/g ANP gel was applied twice a day for 1 week, a slightdegree of erythema appeared, it was possible to see through to thescalp, and the hair became thinner than it had been before application.

Case B20

The test subject was a 50 year old female and was a patient with femalepattern alopecia. The hair of this test subject had thinned from thecrown to the frontal region a few years earlier, and the hair partinghad enlarged. The mother of this test subject had female patternalopecia, and the father had androgenetic alopecia.

When ‘Dermosol-G Lotion’ (Iwaki Seiyaku Co., Ltd.), which contains 1200μg/mL of betamethasone valerate and 1000 μg/mL of gentamicin sulfate,was applied to the crown of this test subject continuously for 8 weeks,the thinning hair was not improved at all.

When 50 μg/g BNP gel was applied to the crown of this test subject twicea day for 1 week, the amount of hair falling out decreased to one thirdafter 1 week, and the thinning hair became inconspicuous after 2 weeks(FIG. 49). After application of the BNP gel was stopped after 2 weeks, astate in which the amount of hair falling out was small was maintainedfor about 1 month, but the amount of hair falling out increased againafter about 1 month.

When 50 μg/g CNP gel was applied twice a day for 2 weeks, in the sameway as for the BNP gel the amount of hair falling out decreased to aboutone third, and the thinning hair became inconspicuous after 2 weeks.

Moreover, when application of the CNP gel was stopped after 2 weeks and2 weeks after that a 50 μg/g CNP:600 μg/mL betamethasone:500 μg/mLgentamicin combination was applied twice a day for 2 weeks, the state inwhich the amount of hair falling out decreased was maintained. Even when2 weeks had elapsed after application of the 50 μg/g CNP:600 μg/mLbetamethasone:500 μg/mL gentamicin combination was stopped after 2weeks, a state in which the thinning hair was inconspicuous and it couldbe said to be normal was maintained.

Case B27

The test subject was a 66 year old female whose hair had thinned atabout 65 years old. This was female pattern alopecia having coexistingseborrheic alopecia with conspicuous seborrheic scale on the scalp. Thistest subject had thinning hair from the crown to the frontal region andthe left-hand side M-shaped part with the skin being quite visible. Withregard to this test subject, the amount of hair falling out was verylarge, and the hair fell out immediately simply by touching it. Thefather and an older brother of this test subject had androgeneticalopecia.

When 50 μg/g BNP gel was applied to the crown of this test subject twicea day, hair growth and hair restoration were observed about 4 to 5 daysafter the application was started, and the seborrheic scale on the scalpwas markedly improved. The amount of hair falling out for this testsubject, for whom a large amount had fallen out simply by touching itbefore the application, decreased. With regard to this test subject, thethinning hair became inconspicuous to a degree such that the skin couldnot be seen 21 days after application of 50 μg/g BNP gel had beenstarted.

22. Therapeutic Effect of CNP Gel on Female Pattern Alopecia

The therapeutic effects of CNP gel on female pattern alopecia are shownin Table 12 (cases C19, C28, and C29).

TABLE 12 Case C19 C28 C29 Figure FIG. 33 FIG. 50 — Gender Female FemaleFemale Age 46 years old 62 years old 60 years old When developed About46 years old 62 years old 59 years old Treatment site Crown Frontalregion and crown Frontal region Hair loss state Diffuse hair thinningwas Hair thinning occurred Hair thinning occurred on conspicuous fromcrown to from frontal region to hairline of frontal frontal region.crown. Large amount of region, accompanied by dandruff on scalp, therescale. was erythema, large amount of hair falling out. Hair loss siteother None None None than head Family history of Grandfather, olderFather had androgenetic Unknown alopecia brother: androgenetic alopeciaat around 35 alopecia years old Family history of Mother: allergicrhinitis Unknown Unknown immune disease Previous history Atopicdermatitis Unknown Unknown Scratch Test Not tested Not tested UnknownEffect of minoxidil Not tested Not tested Unknown Effect of steroid Noeffect Not applied Not applied drug Effect of cooling Not applied Notapplied Not applied therapy Antiallergic drug Not applied Not appliedNot applied Effect of carpronium No effect Not applied Not appliedchloride Effect of Not applied Not applied Not applied cepharanthinDosage form CNP gel CNP gel CNP gel Dose 100 μg/g 50 μg/g 50 μg/g Numberof days used 4 weeks 28 days 14 days Degree of Thinning hair part After2 weeks' Hair thinning at hairline improvement in recovered to almostapplication, hair falling of frontal region symptoms normal state, andhair out decreased, and hair improved to normal state. falling outdecreased. thickened from frontal Scale also disappeared. region tocrown. 1 month after starting application, hair thinning improved tounnoticeable level. Non-recurrence 1 month 3 months 6 weeks periodCase C19

The test subject was a 56 year old female and was a patient with femalepattern alopecia. There was a history of atopic dermatitis, the motherof this test subject was affected by allergic rhinitis, and this was acase with an atopic predisposition. The father of this test subject hadandrogenetic alopecia, and the mother of this test subject had femalepattern alopecia. The results of the scratch test were house dust 2+,mite 2+, cedar 1+, Dactylis 1+, and ragweed 1+. The hair on the crown ofthis test subject had become thin 10 years earlier. This test subjecthad been externally applying a steroid and carpronium chloride, butthere had been no effect.

When 100 μg/g CNP gel was applied to the crown and the thinning hairpart on the crown of this test subject twice a day, the amount of hairfalling out decreased after 4 days of application, new terminal hairgrew on the frontal region and the crown, and the hair parting on thescalp did not widen (ref. FIG. 33). However, when application of the CNPgel was stopped after 2 weeks, about 2 weeks after the application wasstopped a slight degree of erythema and scale reappeared on the scalp ofthe frontal region and the crown, and there was thinning hair.

When this time 50 μg/g BNP gel was applied twice a day for 3 weeks,vellus hair became thick, hair growth was observed, and the thinninghair improved. However, when application of the BNP gel was stoppedafter 3 weeks, about 1 month after the application was stopped a slightdegree of erythema and scale on the scalp of the frontal region and thecrown reappeared, and there was thinning hair.

When 50 μg/g BNP:600 μg/mL betamethasone:500 μg/mL gentamicincombination was applied twice a day for 1 week, vellus hair becamethick, and the thinning hair improved.

Case C28

The test subject was a 62 year old female and was a patient with femalepattern alopecia. This test subject had developed a dry rough scalparound the April following the Great East Japan Earthquake of 11 Mar.2011, and had thinning hair from the frontal region to the crown. Thefather of this test subject had hair loss on the entire head due toandrogenetic alopecia at about 35 years old.

When 50 μg/g CNP gel was applied to the frontal region and the crown ofthis test subject once to twice a day for 2 weeks, the amount of hairfalling out decreased, and marked hair thickening was observed from thefrontal region to the crown. When application of the CNP gel wascontinued for a further 2 weeks, the hair thickened so that thinninghair could hardly be seen (FIG. 50).

1 month after the application was stopped, thinning hair became slightlyconspicuous again, but when a 50 μg/g CNP:600 μg/mL betamethasone:500μg/mL gentamicin combination was applied twice a day for 2 weeks thethickened hair state was maintained.

Case C29

The test subject was a 60 year old female and was a patient with femalepattern alopecia. With regard to this test subject, scale and itchinghad appeared 1 year earlier, and hair at the hair line in the frontalregion had become thin.

When 50 μg/g CNP gel was applied to the frontal region of this testsubject twice a day for 2 weeks, the hair thickened to such a degreethat there was no widening of the hair parting and it was not possibleto see through to the scalp. Even after 6 weeks had elapsed sinceapplication of the CNP gel was stopped, the effects were maintained, andit was not possible to see through to the scalp.

23. Therapeutic Effect of ANP Gel on Seborrheic Alopecia

The therapeutic effects of ANP gel on seborrheic alopecia are shown inTable 13 (cases A11 and A12).

TABLE 13 A11 A12 Case (=A8, B8) (=A9, B11) Figure FIG. 27 Gender MaleMale Age 45 years old 75 years old When developed From 44 years oldAbout 40 s to 50 s Treatment site Crown Crown Hair loss state Thinninghair occurred only on Erythema accompanied by itching and crown, anderythema accompanied seborrheic scale were seen on scalp by itching andseborrheic scale at hair loss site. were seen on scalp at hair losssite. Family history of androgenetic Father: androgenetic alopeciaFather: androgenetic alopecia alopecia Family history of immune NoneNone disease Previous history Atopic dermatitis, allergic Allergicrhinitis rhinitis Scratch Test House dust: 2+ House dust: 2+ Mite: 3+Mite: 2+ Cedar: — Cedar: 3+ Dactylis: 1+ Dactylis: 2+ Ragweed: 1+Ragweed: 2+ Effect of minoxidil Not applied No effect Effect offinasteride Not applied Not applied Effect of glycyrrhetinic acid Notapplied No effect Effect of steroid drug No effect No effect Effect ofcooling therapy Not applied Not applied Antiallergic drug Not appliedNot applied Effect of carpronium chloride Not applied Not applied Effectof cepharanthin Not applied Not applied Dosage form ANP gel ANP gel Dose100 μg/g 100 μg/g Number of days used 2 weeks 4 days Degree ofimprovement in Intense itchiness occurred, No improvement in erythemaand symptoms seborrheic scale aggravated, and seborrheic scale. hairthinning range enlarged. Non-recurrence period No improvement NoimprovementCase A11

Case A11 was a case with coexisting androgenetic alopecia and seborrheicalopecia and is the same case as cases A8 and B8. Details of this caseare as described for cases A8 and B8; when 100 μg/g ANP gel was appliedto the entire hair loss site of the crown twice a day morning andevening, intense itching appeared on the 2^(nd) day, the amount of hairfalling out increased, the hair loss range enlarged, seborrheic scalpwas aggravated, erythema appeared, and no hair growth was observed.

Case A12

Case A12 was a case with coexisting androgenetic alopecia and seborrheicalopecia and is the same case as cases A9 and B11. Details of this caseare as described for cases A9 and B11; when 100 μg/g ANP gel was appliedto the hair loss site of the crown twice a day morning and evening for 4days, there was no improvement in erythema and seborrheic scale, andthere was no improvement effect for alopecia either.

24. Therapeutic Effect of BNP Gel on Seborrheic Alopecia

The therapeutic effects of BNP gel on seborrheic alopecia are shown inTable 14 (cases B21 and B22).

TABLE 14 B21 B22 Case (=A8, B8) (=A9, B11) Figure Gender Male Male Age45 years old 75 years old When developed From 44 years old 40 sTreatment site Crown Crown Hair loss state Hair thinning occurred onlyon Hair thinning occurred on crown, crown, and erythema accompanied anderythema accompanied by itching by itching and seborrheic scale andseborrheic scale were seen on were seen on scalp at hair loss scalp athair loss site. site. Family history of androgenetic Father:androgenetic alopecia Father: androgenetic alopecia alopecia Familyhistory of immune None None disease Previous history Atopic dermatitis,allergic Allergic rhinitis rhinitis Scratch Test House dust: 2+ Housedust: 2+ Mite: 3+ Mite: 2+ Cedar: — Cedar: 3+ Dactylis: 1+ Dactylis: 2+Ragweed: 1+ Ragweed: 2+ Effect of minoxidil Not applied No effect Effectof finasteride Not applied Not applied Effect of glycyrrhetinic acid Notapplied No effect Effect of steroid drug No effect No effect Effect ofcooling therapy Not applied Not applied Antiallergic drug No effect Noeffect Effect of carpronium chloride No effect Not applied Effect ofcepharanthin Not applied Not applied Dosage form BNP gel BNP gel Dose 50μg/g 100 μg/g Number of days used 5 weeks 3 weeks Degree of improvementin Erythema, seborrheic scale, and Erythema, seborrheic scale, andsymptoms itching at hair loss site itching at hair loss site markedlymarkedly improved and hair improved, hair falling out thinning improved.decreased, and hair quality improved and became dark. Non-recurrenceperiod Treatment continuing Treatment continuingCase B21

Case B21 was a case with coexisting androgenetic alopecia and seborrheicalopecia and is the same case as cases A8 and B8. Details of this caseare as described for cases A8 and B8.

When 50 μg/g BNP gel was applied to the entire hair loss site of thecrown of this test subject twice a day morning and evening, on the1^(st) week after application of the BNP gel was started erythema,seborrheic scale, and itching on the hair loss site markedly improved,the amount of hair falling out decreased to an unnoticeable level, hairbecame thick on the crown objectively, the number of terminal hairsincreased, and the thinning hair improved.

When application of the BNP gel to this test subject was stopped after 3weeks, the feeling of volume on the crown decreased after 2 months, anditching started.

When multilayer application to the crown was carried out with 5%minoxidil solution and then with 100 μg/g BNP gel, there were nosymptoms of irritation as there had been when the ANP gel was applied,when 1 week had elapsed after the multilayer application was started theamount of hair falling out decreased, the itchiness disappeared, andhair growth effects were observed. This test subject is still continuingto receive multilayer application with 5% minoxidil solution and 100μg/g BNP gel at the present.

Case B22

Case B22 was a case with coexisting androgenetic alopecia and seborrheicalopecia and is the same as cases A9 and B11. Details of this case areas described for cases A9 and B11.

When 100 μg/g BNP gel was applied to thinning hair sites of the crownand the M-shaped part of this test subject twice a day morning andevening, on the 3^(rd) day after the application was started erythema,seborrheic scale, and itching in the hair loss sites markedly improved,and the amount of hair falling out become unnoticeable. With regard tothis test subject, 2 weeks after, the hair quality improved, the hairbecame thick and dark, and there was hair thickening. With regard tothis test subject, even in the M-shaped part, in the 2^(nd) week ofapplication vellus hair became thick and dark, and the hair became long,albeit slowly.

In order to examine an external preparation that could further reducethe thinning hair range of the crown of this test subject, after a 5%minoxidil solution was applied to the crown and the M-shaped part, 100μg/g BNP gel was applied by multilayer application, compared withapplication of a single BNP agent, the hair on the crown became blackand dark when 1 week had elapsed since the multilayer application hadstarted, and there was hair thickening. This test subject is stillhaving multilayer application treatment with 5% minoxidil and 100 μg/gBNP gel at the present.

25. Therapeutic Effect of ANP Gel on Alopecia Pityroides

The therapeutic effects of ANP gel on alopecia pityroides are shown inTable 15 (case A13).

TABLE 15 A13 A14 Case (=A6, C3, C4) (=A1, C5) Figure FIG. 21 GenderFemale Female Age 50 years old 33 years old When developed 40 years old13 years old Hair loss range S2 S3 Treatment site Crown Right temporalregion Hair loss site other than head None (B0) Eyebrows (B1) Familyhistory of alopecia Mother: alopecia areata None Family history ofimmune Child: allergic rhinitis, Mother: atopic dermatitis diseasechronic urticaria Older sister: atopic dermatitis, allergic rhinitisPrevious history Atopic dermatitis, allergic Atopic dermatitis rhinitisScratch Test House dust: 1+ House dust: 2+ Mite: 1+ Mite: 1+ Cedar: —Cedar: — Dactylis: 2+ Dactylis: 2+ Ragweed: 1+ Ragweed: 2+ Effect ofminoxidil Not applied Not applied Effect of finasteride ContraindicatedContraindicated Effect of glycyrrhetinic acid Not applied Not appliedEffect of steroid drug No effect No effect Effect of cooling therapy Notapplied No effect Antiallergic drug No effect No effect Effect ofcarpronium chloride No effect Not applied Effect of cepharanthin Notapplied Not applied Dosage form ANP gel ANP ointment Dose 100 μg/g 100μg/g Number of days used 14 days 21 days Degree of improvement in S2→S2S3→S3 symptoms Non-recurrence period — —Case A13

Case A13 was a case with coexisting alopecia areata and alopeciapityroides and is the same case as cases A6, C3, C4, and C30. CNPointment was applied to the crown. Details of this case are as describedfor cases A6, C3, and C4; there was no effect from application of 100μg/g ANP gel for 2 weeks, dry rough scale increased, there was itching,and the hair loss range enlarged somewhat.

26. Therapeutic Effect of ANP Ointment on Alopecia Pityroides

The therapeutic effects of ANP ointment on alopecia pityroides are shownin Table 15 (case A14).

Case A14

Case A14 was a case with coexisting alopecia areata and alopeciapityroides and is the same case as cases A1 and C5. It should be notedthat CNP ointment was applied after a 2 week period of drug suspension.Details of this case are as described for cases A1 and C5; when 100 μg/gANP gel was applied twice a day for 3 weeks, there was growth of vellushair, but erythema and pityriatic desquamation on the scalp were notalleviated, and there was itching.

27. Therapeutic Effect of BNP Gel on Alopecia Pityroides

The therapeutic effects of BNP gel on alopecia pityroides are shown inTable 16 (cases B23 and B28).

TABLE 16 Case B23 B28 Figure FIG. 51 FIG. 53 Gender Male Male Age 81years old 59 years old When developed About 78 years old 57 years oldTreatment site Frontal region and crown Frontal region and crown Hairloss state Hair thinning was conspicuous Frontal region and crown allover Hair loss site other than head None None (B0) Family history ofalopecia None None Family history of immune None None disease Previoushistory None None Scratch Test Not tested Negative Effect of minoxidilNot applied No effect Effect of finasteride Not applied Not appliedEffect of glycyrrhetinic acid Not applied Not applied Effect of steroiddrug Not applied Not applied Effect of cooling therapy Not applied Notapplied Antiallergic drug Not applied Not applied Effect of carproniumchloride Not applied Not applied Effect of cepharanthin Not applied Notapplied Dosage form BNP gel BNP gel Dose 50 μg/g, 200 μg/g 20 μg/gNumber of days used 14 days + 5 days 7 days Degree of improvement inPityriatic scale improved, Dandruff, scale, and itching symptoms vellushair turned into terminal disappeared, and hair falling out hair andbecame thick and black, decreased. Hair growth and hair and thinninghair improved. thickening were observed. Non-recurrence period —Treatment continuingCase B23

The test subject was a 81 year old male who had developed thinning hairfrom the frontal region to the crown 3 years earlier. This was a casewith coexisting senile alopecia and alopecia pityroides in whichpityriatic scale was seen on the scalp.

After 50 μg/g BNP gel was applied to the frontal region and the crown ofthis test subject for 2 weeks and suspended for 1 week, when 200 μg/gBNP gel was applied once a day for 5 days, pityriatic desquamation wasalleviated, vellus hair became terminal hair and black, and thinninghair was greatly improved (FIG. 51).

Case B28

The test subject was a 59 year old male and had alopecia pityroides.This test subject was in a thinning hair state in which there was hardlyany hair from the frontal region to the crown. Thinning hair hadoccurred 2 years earlier, RiUP X5 (trademark), which contains 0.05 g/mLminoxidil, had been applied twice a day for one and a half years, butdandruff had became worse, itchiness had occurred, and the amount ofhair falling out and the thinning hair had rapidly become aggravated 4months earlier. This test subject was in a state in which a large amountof hair became attached to scale on the scalp and fell out when washingthe hair.

When 20 μg/g BNP gel was applied to the frontal region and the crown ofthis test subject twice a day for 7 days, the effect was very good;there was no itching, dandruff stopped occurring, and the amount of hairfalling out when washing the hair decreased to a few hairs. Inaccordance with application of the BNP gel, scale thickly deposited onthe frontal region and the crown disappeared, and quite clear hairgrowth and hair thickening were observed from the frontal region to thecrown (FIG. 53).

28. Therapeutic Effect of CNP Gel on Alopecia Pityroides

The therapeutic effects of CNP gel on alopecia pityroides are shown inTable 17 (cases C30 and C31).

TABLE 17 C30 Case (=A6, A13, C3, C4, C33) C31 Figure FIG. 52 GenderFemale Male Age 50 years old 42 years old When developed 40 years oldLate 30s Hair loss range S2 Frontal region and crown Treatment site Lefttemporal region Frontal region and crown Hair loss site other than headNone (B0) None (B0) Family history of alopecia Mother: alopecia areataFather: androgenetic alopecia Family history of immune Child: allergicrhinitis, Younger brother: atopic dermatitis disease chronic urticariaPrevious history Atopic dermatitis, allergic None rhinitis Scratch TestHouse dust: 1+ House dust: 3+ Mite: 1+ Mite: 3+ Cedar: — Cedar: 2+Dactylis: 2+ Dactylis: 2+ Ragweed: 1+ Ragweed: 1+ Effect of minoxidilNot applied Not applied Effect of finasteride Contraindicated Notapplied Effect of glycyrrhetinic acid Not applied Not applied Effect ofsteroid drug No effect No effect on hair falling out or dandruff after 2years′ external application. Effect of cooling therapy Not applied Notapplied Antiallergic drug No effect Not applied Effect of carproniumchloride No effect Not applied Effect of cepharanthin Not applied Notapplied Dosage form CNP gel CNP gel Dose 100 μg/g 100 μg/g Number ofdays used 21 days 14 days Degree of improvement in S2→S0 Thick scalemarkedly improved and symptoms hair thickened. Non-recurrence period 8months Treatment continuingCase C30

Case C30 was a case with coexisting alopecia areata and alopeciapityroides and is the same case as cases A6, C3, C4, and A13. It shouldbe noted that CNP ointment was applied to the right frontal region. Thedetails of this case are as described for cases A6, C3, and C4; withapplication of 100 μg/g CNP gel twice a day for 3 weeks, there was goodprogress even after application was stopped, and there was no recurrenceat the application site even when 8 months had elapsed after theapplication was stopped.

Case C31

The test subject was a 42 year old male and was a patient with alopeciapityroides. From the late 30s the hair of the frontal region and thecrown had become thin. From 2 years earlier, ‘Nitrazen Cream 2% forexternal application’ (Iwaki Seiyaku Co., Ltd.), which contains 2%ketoconazole, which is an antifungal agent, and ‘Betnoval G OintmentCream’ (Sato Pharmaceutical Co., Ltd.), which contains 1.2 mg/gbetamethasone valerate, which is a synthetic adrenal cortex hormone, and1 mg/g of gentamicin, which is an antibiotic, had been appliedcontinuously, but not only were there no effects at all, but alsodandruff had adhered thickly and the thinning hair had become aggravatedfrom 1 year earlier.

When 100 μg/g CNP gel was applied to the frontal region and the crown ofthis test subject twice a day for 2 weeks, scale that had been thicklyadhering disappeared considerably, and hair started to thicken on thecrown (FIG. 52).

29. Therapeutic Effect of CNP Ointment on Alopecia Pityroides

The therapeutic effects of CNP ointment on alopecia pityroides are shownin Table 18 (case C33).

TABLE 18 Case C33 (=A6, A13, C3, C4, C30) Figure Gender Female Age 50years old When developed 40 years old Hair loss range S2 Treatment siteRight frontal region Hair loss site other than head None (B0) Familyhistory of alopecia Mother: alopecia areata Family history of immunedisease Child: allergic rhinitis, chronic urticaria Previous historyAtopic dermatitis, allergic rhinitis Scratch Test House dust: 1+ Mite:1+ Cedar: — Dactylis: 2+ Ragweed: 1+ Effect of minoxidil Not appliedEffect of finasteride Contraindicated Effect of glycyrrhetinic acid Notapplied Effect of steroid drug No effect Effect of cooling therapy Notapplied Antiallergic drug No effect Effect of carpronium chloride Noeffect Effect of cepharanthin Not applied Dosage form CNP ointment Dose100 μg/g Number of days used 14 days Degree of improvement in symptomsS2→S1 Non-recurrence period 1 yearCase C33

Case C33 was a case with coexisting alopecia areata and alopeciapityroides and is the same case as cases A6, C3, C4, A13, and C30. Itshould be noted that CNP ointment was applied to the right frontalregion. The details of this case are as described for cases A6, C3, andC4, and by applying 100 μg/g CNP ointment twice a day for 2 weeks clearhair growth was observed and the erythema and scale on the scalpdisappeared.

30. Therapeutic Effect of ANP Gel on Senile Alopecia

The therapeutic effects of ANP gel on senile alopecia are shown in Table19 (case A15).

TABLE 19 Case A15 Figure FIG. 54 Gender Male Age 74 years old Whendeveloped Unknown Treatment site Frontal region and crown Hair lossstate Hair thinning was conspicuous all over. Hair loss site other thanhead None Family history of alopecia None Family history of immunedisease None Previous history None Scratch Test Not tested Effect ofminoxidil Not applied Effect of finasteride Not applied Effect ofglycyrrhetinic acid Not applied Effect of steroid drug Not appliedEffect of cooling therapy Not applied Antiallergic drug Not appliedEffect of carpronium chloride Not applied Effect of cepharanthin Notapplied Dosage form ANP gel (CNP gel) Dose ANP gel: 100 μg/g (CNP gel:50 μg/g) Number of days used 1 week Degree of improvement in symptomsErythema, scale, and itching aggravated, no hair thickening effect, andhair loss increased somewhat. Non-recurrence period Treatment continuingCase A15

The test subject was a 74 year old male who had senile alopecia in whichthere was dandruff, there was a large amount of hair falling out, therewas thinning hair over the entire head part, and white hair wasconspicuous. This test subject had intense itchiness on the scalp, theamount of dandruff was large, and the amount of hair falling out waslarge.

When 100 μg/g ANP gel was applied to the crown of this test subjecttwice a day for 1 week, erythema, scale, and itching were aggravated,there was no hair thickening effect, and the hair loss increasedsomewhat (FIG. 54).

When ANP gel was stopped after 1 week and from the following day 50 μg/gCNP gel was applied twice a day for 2 days, erythema, scale, and itchingwere alleviated. For a further 4 weeks after that, 50 μg/g CNP gelcontinued to be applied twice a day, clear hair thickening was observedin the frontal region in particular, and hair stopped falling out (FIG.54). Hair that had grown was black terminal hair and did not fall outeven when rubbed strongly. It was surprising to observe the hairthickening effect of the CNP gel for such an old age of 74.

31. Therapeutic Effect of BNP Gel on Senile Alopecia

The therapeutic effects of BNP gel on senile alopecia are shown in Table20 (B24).

TABLE 20 Case B24 Figure FIG. 51 Gender Male Age 81 years old Whendeveloped About 78 years old Treatment site Frontal region and crownHair loss state Hair thinning was conspicuous all over. Hair loss siteother than head None Family history of alopecia None Family history ofimmune disease None Previous history None Scratch Test Not tested Effectof minoxidil Not applied Effect of finasteride Not applied Effect ofglycyrrhetinic acid Not applied Effect of steroid drug Not appliedEffect of cooling therapy Not applied Antiallergic drug Not appliedEffect of carpronium chloride Not applied Effect of cepharanthin Notapplied Dosage form BNP gel Dose 50 μg/g, 200 μg/g Number of days used14 days + 5 days Degree of improvement in symptoms Pityriatic scaleimproved, vellus hair turned into terminal hair and became thick andblack, and hair thinning improved. Non-recurrence period TreatmentcontinuingCase B24

Case B24 was a case with coexisting senile alopecia and alopeciapityroides, and is the same case as case B23. Details of this case areas described for case B23.

When 200 μg/g BNP gel was applied to the frontal region and the crown ofthis test subject once a day for 5 days, pityriatic desquamation wasalleviated, rich black terminal hair grew, and the thinning hair wasgreatly improved (FIG. 51). Most of the hair was white before theapplication, but hair that grew after the application was black.

32. Therapeutic Effect of CNP Gel on Senile Alopecia

The therapeutic effects of CNP gel on senile alopecia are shown in Table21 (case C34).

TABLE 21 Case C34 Figure Gender Female Age 70 years old When developedAbout 68 years old Treatment site Crown Hair loss state Hair thinningwas conspicuous from frontal region to crown. Hair loss site other thanhead None Family history of alopecia None Family history of immunedisease None Previous history None Scratch Test Not tested Effect ofminoxidil Not applied Effect of finasteride Not applied Effect ofglycyrrhetinic acid Not applied Effect of steroid drug No effect Effectof cooling therapy Not applied Antiallergic drug Not applied Effect ofcarpronium chloride Not applied Effect of cepharanthin Not appliedDosage form CNP gel Dose 50 μg/g Number of days used 7 days Degree ofimprovement in symptoms Hair became supple, and thinning hair on crownimproved to unnoticeable level. Hair falling out decreased, and therewas no pain on scalp during external application. Non-recurrence period2 weeksCase C34

The test subject was a 70 year old female and had thinning hair on thecrown from 68 years old. According to the test subject, the skin of thecrown with the thinning hair became atrophic and was too painful totouch. When 50 μg/g CNP gel was applied twice a day for 3 days, the painof the scalp was alleviated, and the amount of hair falling outdecreased. After that, when the application was continued for a further3 days, erythema of the scalp disappeared, itching and pain becameunnoticeable, and there was hair thickening.

When the medication was changed to only ‘Dermosol G lotion (Dermosol-GLotion)’ (Iwaki Seiyaku Co., Ltd.), which contains 1200 μg/mLbetamethasone valerate and 1000 μg/mL gentamicin sulfate, the skin ofthe crown started tingling after 3 days, a slight degree of erythemareappeared, and there was thinning hair.

When 100 μg/g BNP gel was applied once a day for 1 week, the erythema onthe crown disappeared, the hair thickened, and the thinning hair becameinconspicuous.

When a 50 μg/g BNP:600 μg/mL betamethasone:500 μg/mL gentamicincombination was applied for 1 week, the symptoms of erythema on thecrown and thinning hair, which were exhibited with application ofDermosol G lotion alone, did not appear, and a state with hairthickening was maintained even when 3 weeks had elapsed after theapplication was started.

33. Therapeutic Effect of BNP Gel on Cancer Chemotherapy Drug-InducedAlopecia

The therapeutic effects of BNP gel on cancer chemotherapy drug-inducedalopecia are shown in Table 22 (case B25).

TABLE 22 Case B25 Figure Gender Female Age 54 years old When developed54 years old Cancer chemotherapy R-CHOP therapy Treatment site Hairlinepart in frontal region Hair loss state White hair all over. Left-handside hairline part retreated and had hair thinning. Hair loss site otherthan head Whole body Family history of alopecia None Family history ofimmune disease None Previous history None Scratch Test Not tested Effectof minoxidil Not applied Effect of glycyrrhetinic acid Not appliedEffect of steroid drug Not used Effect of cooling therapy Not appliedAntiallergic drug Not applied Effect of carpronium chloride Not appliedEffect of cepharanthin Not applied Dosage form BNP gel Dose 100 μg/gNumber of days used 7 days Degree of improvement in symptoms Growth andrestoration of black terminal hair was observed in left front temporalregion, which had white hair and was thin, after 2 weeks. Non-recurrenceperiod 3 monthsCase B25

The test subject was a 54 year old female. The test subject wasdiagnosed with malignant lymphoma and had received 6 courses of R-CHOPtherapy from April to September 2011. The R-CHOP therapy referred tohere is one type of cocktail therapy using a plurality of chemotherapydrugs in which 1 course of the administration schedule consisted of anintravenous drip of rituximab, which is a mouse-human•chimericmonoclonal antibody against CD20, which is a human B cell surfaceantigen, on the 1^(st) day, oral administration, after each meal, ofprednisolone tablet, which is a synthetic adrenal cortex hormone drug,intravenous injection of vincristine, which is a microtubule inhibitor,intravenous injection of doxorubicin, which is an inhibitor of DNA andRNA synthesis, and intravenous injection of cyclophosphamide, which is aprodrug of an inhibitor of DNA synthesis, on the 2^(nd) day, only oraladministration, after each meal, of prednisolone tablet from the 3^(rd)day to the 6^(th) day, and a drug suspension period from the 7^(th) dayto the 21^(st) day, and this administration cycle is repeated. Withregard to the test subject, the hair started to fall out from the 2^(nd)week of the first course, and all the hair had fallen out in July whenthe 4^(th) course had ended. When the treatment was completed inSeptember, the hair started to grow gradually; the hair that grew waspure white, and the left-hand side hair line part had retreated and hadbecome very thin.

When 100 μg/g BNP gel was applied to this left-hand side hair line parttwice a day for 1 week, black terminal hair started to grow in thethinning hair part with white downy hair after 2 weeks, the hairgradually thickened, and black hair seemed to have grown in a roundshape in the hair line part that had retreated in an M-shape. In thesite to which the BNP gel had been applied in the left-hand side hairline M-shaped site, all the hair that had grown was black. Although noapplication was carried out for 3 months after that hair did not fallout again and there was no formation of white hair.

34. Therapeutic Effect of CNP Gel on Cancer Chemotherapy Drug-InducedAlopecia

The therapeutic effects of BNP gel on cancer chemotherapy drug-inducedalopecia are shown in Table 23 (case C35).

TABLE 23 Case C35 Figure FIG. 56-1, FIG. 56-2 Gender Female Age 47 yearsold When developed 47 years old Cancer chemotherapy Cisplatin Treatmentsite Frontal region, crown Hair loss state Hair thinning was conspicuousfrom frontal region to crown, mainly with fine vellus hair. Hair losssite other than head Whole body Family history of alopecia UnknownFamily history of immune disease Child: atopic dermatitis Previoushistory Cancer of body of uterus, alopecia areata, atopic dermatitis,allergic rhinitis Scratch Test House dust: 3+ Mite: 3+ Cedar: —Dactylis: — Ragweed: 1+ Effect of minoxidil Not applied Effect ofglycyrrhetinic acid Not applied Effect of steroid drug No effect Effectof cooling therapy Not applied Antiallergic drug No effect Effect ofcarpronium chloride No effect Effect of cepharanthin No effect Dosageform CNP gel Dose 50 μg/g Number of days used 6 weeks Degree ofimprovement in symptoms Hair thickened, and hairs became thick and dark,and was restored. Non-recurrence period Treatment continuingCase C35

The test subject was a 47 year old female. The test subject had receivedan operation to remove cancer of the body of the uterus in November2009, had 6 courses of treatment with cisplatin, which is a cancerchemotherapy drug, and had completed the cancer chemotherapy in March2010. The test subject showed hair loss of the entire head accompanyingthe treatment with cisplatin, and although fine soft short hair grewaround May 2011 when almost 1 year had elapsed since the treatment withcisplatin was completed, an area from the frontal region to the crownrecovered only to a state with thin hair. There had been no effect onthe test subject when carpronium chloride had been applied thereto for 6months.

The application of carpronium chloride was therefore stopped. When, 2months after stopping, 50 μg/g CNP gel was applied twice a day for 6weeks to the area from the frontal region to the crown, the number ofhairs increased and the hair thickened considerably from the frontalregion to the crown, and the hair became thick and long (FIG. 56-1).

When application of the CNP gel was stopped after 6 weeks, and 3 weeksafter that a CNP:betamethasone:gentamicin combination was applied twicea day for 2 weeks, the hair thickened further.

At this point, new alopecia areata occurred in the left temporal region;a 50 μg/g CNP:600 μg/mL betamethasone:500 μg/mL gentamicin combinationwas applied twice a day, hair growth was confirmed after 2 weeks, andmarked growth of terminal hair was confirmed after 3 weeks (FIG. 56-2).

Conclusions from Case Test Results

As is clear from the above-mentioned test cases, the agent for thetreatment of alopecia of the present invention had a very high recoveryrate for hair loss, and the period taken to express its hair growthpromoting effect was short. In most cases, hair growth was confirmed byapplying the agent for the treatment of alopecia of the presentinvention for 1 week to 2 weeks, and clear hair growth was observedduring the 3^(rd) week. Furthermore, the treatment agent of the presentinvention restored white hair to black hair, decreased dandruff in analopecia pityroides patient, and improved seborrheic scalp in aseborrheic alopecia patient.

When the agent for the treatment of alopecia of the present inventioncontained CNP or BNP as an active ingredient, the therapeutic effectswere marked, hair grew almost certainly with application twice a day for1 week, terminal hair was observed with application for 2 weeks, and itbecame difficult to see the skin with application for 4 weeks. Worthy ofspecial note is that it was unnecessary to continue application aftervellus hair grew; the vellus hair became dark and thick, became terminalhair, and continued to grow.

In particular, when the agent for the treatment of alopecia of thepresent invention contained CNP as an active ingredient it could morereliably suppress inflammation of a hair loss site than one containingBNP as an active ingredient, the amount of hair falling out decreaseddramatically in 1 day to a few days, and hair grew earlier. On the otherhand, the agent for the treatment of alopecia of the present inventioncontaining BNP as an active ingredient was characterized by blackterminal hair often growing. Furthermore, when the agent for thetreatment of alopecia of the present invention contained ANP as anactive ingredient, it was effective only for alopecia in which there wasno erythema, scale, seborrheic desquamation, etc. on the scalp, butthere were many cases in which erythema and itchiness occurred and thestate became worse than that before application.

Moreover, the agent for the treatment of alopecia of the presentinvention exhibited marked effects on alopecia areata and androgeneticalopecia, could suppress inflammation of seborrheic alopecia andalopecia pityroides so that hair could grow, dramatically decreaseddandruff, and could suppress itchiness. Furthermore, it promotedrestoration of hair in female pattern alopecia, postpartum alopecia, andsenile alopecia, and it was also confirmed that it promoted growth ofblack hair in cancer chemotherapy drug-induced alopecia.

The agent for the treatment of alopecia of the present inventionexhibited an improvement effect with long duration for androgeneticalopecia, female pattern alopecia, seborrheic alopecia, alopeciapityroides, and senile alopecia, and there was no recurrence of hairloss at the application site for at least 2 months after the applicationwas stopped.

These therapeutic effects of the present invention in the treatment ofandrogenetic alopecia were in marked contrast to the fact that in agroup that has had orally administered finasteride for 1 year and hasthen stopped administration, the improvement effect disappears andandrogenetic alopecia progresses. That is, the effect of finasteride isseen only while it is being orally administered, whereas with externalapplication of the agent for the treatment of alopecia of the presentinvention for 1 to 3 weeks, the improved hair growth state could bemaintained for about 2 months after its use was stopped. After 3 monthshad elapsed since application of the agent for the treatment of alopeciaof the present invention was stopped, there was a case in which theoriginal hair loss state returned, but even in this case reapplying theagent for the treatment of alopecia of the present invention allowed thesame black terminal hair to grow as previously without side effects.

Moreover, with regard to alopecia areata and cancer chemotherapydrug-induced alopecia, with external application of the agent for thetreatment of alopecia of the present invention for 1 to 3 weeks, thehair growth state kept improving even after its use was stopped andthere was a cure, and there was no recurrence of alopecia at theapplication site.

Moreover, BNP and CNP did not exhibit any adverse events such as localsymptoms of irritation, skin atrophy, or an itching sensation resultingfrom their application, and there were no systemic side effects.

Since it was confirmed that the agent for the treatment of alopecia ofthe present invention exhibited marked effects in the treatment ofalopecia areata, androgenetic alopecia, seborrheic alopecia, alopeciapityroides, female pattern alopecia, postpartum alopecia, senilealopecia, and cancer chemotherapy drug-induced alopecia, it wassubstantially proved that it was effective not only for the treatment ofthe above but also for the treatment of almost all types of alopecia.

That is, since the above-mentioned respective types of alopecia havecompletely different development mechanisms from each other, beingeffective for these various different types of alopecia clearly meansbeing useful for the treatment of all types of alopecia.

Furthermore, as is disclosed in the present specification, since thetest subjects for whom the effectiveness of the agent for the treatmentof alopecia of the present invention had been confirmed include manytest subjects both with and without a history of, or coexisting, immunedisease, and test subjects having a biological family both with andwithout a history of, or coexisting, immune disease, the agent for thetreatment of alopecia of the present invention is useful for targetsboth with and without a genetic background or a history of, orcoexisting, immune disease. Furthermore, with regard to the relationshipbetween the test subjects and an allergic predisposition, there was nosystematic relationship between the effectiveness of the agent for thetreatment of alopecia of the present invention and the results of thescratch test of the test subjects for whom the effectiveness wasconfirmed; the agent for the treatment of alopecia of the presentinvention is effective for targets both with and without an allergicpredisposition, regardless of the presence or absence of an allergicpredisposition.

It was confirmed that the agent for the treatment of alopecia of thepresent invention has a marked effect on the treatment of alopeciaareata and androgenetic alopecia. The period taken to confirm hairgrowth subjectively was short not only for alopecia areata monolocularisbut also even for intractable alopecia areata multilocularis, in whichthe hair loss period is long and the hair loss range is wide, and evenfor intractable alopecia areata ophiasis; there is no pain accompanyingthe treatment, it is therefore possible to recover from emotionaldistress and regain QOL (quality of life) at an early stage, and it canbe said to be a completely new therapy that can be highly recommended.

It is clear that the agent for the treatment of alopecia of the presentinvention is very effective not only for alopecia areata monolocularisbut also for S2 or greater alopecia areata multilocularis, which issevere, a case in which hair loss occurs in another place in addition tothe head, a case with a coexisting atopic disease, which is said to beintractable, and alopecia ophiasis.

The agent for the treatment of alopecia of the present invention couldrestore the hair of a patient with postpartum alopecia that did not curespontaneously even over half a year after childbirth to the same stateas that before childbirth, and it became clear that it was effective fora target having resistance to treatment with a steroid or carproniumchloride.

The agent for the treatment of alopecia of the present invention wasalso effective for female pattern alopecia and could restore the hair tosuch an extent that it became unnoticeable in appearance with 1 to 2weeks' application, and it was effective for a patient having resistanceto treatment with carpronium chloride, a steroid, and an antifungalagent. It was confirmed that, when the agent for the treatment ofalopecia of the present invention was used for female pattern alopecia,there was a case in which thinning hair reoccurred about 1 month afterthe application was stopped, but re-application thereof could restorethe hair in the same manner as that of the previous application, and noside effects were seen.

It became clear that the agent for the treatment of alopecia of thepresent invention could markedly improve erythema, seborrheic scale, anditching of a hair loss site of seborrheic alopecia and decrease theamount of hair falling out.

It was confirmed that the agent for the treatment of alopecia of thepresent invention could make erythema and scale of alopecia pityroidesdisappear, make black terminal hair grow, and prevent recurrence evenwhen 1 month had elapsed after the application was stopped. Furthermore,the agent for the treatment of alopecia of the present inventionexhibits marked effects on alopecia pityroides having resistance totreatment with an antifungal agent or a steroid.

It was shown that the agent for the treatment of alopecia of the presentinvention could relieve pain and itching of the scalp in senilealopecia, alleviate erythema and pityriatic desquamation, and make blackterminal hair grow.

It was confirmed that the agent for the treatment of alopecia of thepresent invention was effective for cancer chemotherapy drug-inducedalopecia and could make black hair grow even in an area in which hairhad become white and maintain a hair growth state even when 2 months orlonger had elapsed after the application was stopped.

The agent for the treatment of alopecia of the present inventionexhibited a very marked therapeutic effect for alopecia if either of BNPor CNP was an active ingredient. Furthermore, when there was noerythema, scale, seborrheic erythema, seborrheic desquamation, orpityriatic desquamation on the scalp, one containing ANP as an activeingredient exhibited a hair growth effect.

Therefore, it can be appreciated that a chimeric peptide of 2 or moreNPs selected from ANP, BNP, and CNP also exhibits a therapeutic effectfor alopecia. Moreover, when taking into consideration that it isgenerally thought that ANP and BNP activate the NPR-A receptor so as tobring about vasodilatory action, diuretic action, and cytostatic action,and CNP shows a growth-inhibitory action on vascular smooth muscle cellsvia the NPR-B receptor, it is surprising that the agent for thetreatment of alopecia of the present invention containing CNP or BNP asan active ingredient exhibited a particularly marked therapeutic effectfor alopecia, and it is sufficient to appreciate that a chimeric peptideof BNP and CNP exhibits the same therapeutic effect for alopecia.

Alopecia areata occurs in young females at the same frequency as inmales, and in spite of the appearance being greatly impaired, there areonly rough treatment methods involving local injection of a steroid orintentionally irritating the skin in anticipation of producing immunemodulation.

Moreover, the therapeutic effects of the above methods are not high, andthere might be adverse events such as skin atrophy due to steroid localinjection or systemic contact dermatitis from local immunotherapy. Onthe other hand, the agent for the treatment of alopecia of the presentinvention exhibits remarkable effects on alopecia areata in particular,there are no side effects from BNP and CNP, the effects are not onlyseen during application but the improved state is also maintained afterapplication is stopped, and although a new bald area might occur at asite to which no application is made, there was no recurrence in theapplication sites as far as the cases that have been experienced areconcerned, and this is great hope for a patient suffering from alopeciaareata.

INDUSTRIAL APPLICABILITY

The agent for the treatment of alopecia of the present inventioncontaining a natriuretic peptide (NP) as an active ingredient promoteshair regeneration, hair growth, and hair thickening on a hair loss siteof an alopecia areata patient, an androgenetic alopecia patient, afemale pattern alopecia patient, a postpartum alopecia patient, aseborrheic alopecia patient, an alopecia pityroides patient, a senilealopecia patient, a cancer chemotherapy drug-induced alopecia patient,and a patient with alopecia due to radiation exposure, can improve thealopecia outstandingly, does not cause side effects such as an itchingsensation, irritation, or feminization, and the therapeutic effects foralopecia are not lost over a long period even when its use is stopped.

Furthermore, it shows therapeutic effects on androgenetic alopecia thatexhibits resistance to treatment with minoxidil or finasteride, andshows marked therapeutic effects on alopecia areata that exhibitsresistance to treatment with steroid local injection or localimmunotherapy.

Therefore, the agent for the treatment of alopecia of the presentinvention can be anticipated to be useful as a very effective treatmentdrug for alopecia for which sufficient therapeutic effects cannot beobtained by the conventional minoxidil or finasteride, and alopecia thatdevelops in relation to an immune overreaction or an immune abnormality.

In particular, the agent for the treatment of alopecia of the presentinvention can dramatically improve severe alopecia on the adult headthat has been very difficult to treat and causes problems in sociallife, without any side effects at all. The agent for the treatment ofalopecia of the present invention is not only effective for intractablealopecia but also exhibits the same effects regardless of gender and isnot limited to adults but is also effective for patients in their teens.

Therefore, practical use of the agent for the treatment of alopecia ofthe present invention as a new agent for the treatment of androgeneticalopecia that replaces minoxidil or finasteride can be anticipated, andthe practical use thereof as an agent for the treatment of alopeciaareata, which has had no effective therapy for a long time, is verypromising.

[Sequence Listing]

The invention claimed is:
 1. A method of treating alopecia comprising:externally applying a composition that comprises an effective amount ofC-type natriuretic peptide (CNP) to a required site of skin of a subjectin need of such treatment, wherein the CNP comprises an amino acidsequence selected from the group comprising the human amino acidsequence of CNP-22 or CNP-53, and wherein the alopecia is one or moreselected from the group consisting of alopecia areata, androgeneticalopecia, seborrheic alopecia, alopecia pityroides, female patternalopecia, postpartum alopecia, senile alopecia, cancer chemotherapydrug-induced alopecia and alopecia due to radiation exposure.
 2. Themethod of treating alopecia according to claim 1, wherein thecomposition further comprises an agent for the treatment of white hair,an agent for the treatment of vellus hair formation, or an agent forinhibiting seborrheic scalp or the occurrence of dandruff.
 3. The methodof treating alopecia according to claim 1, wherein the androgeneticalopecia is androgenetic alopecia in a male or androgenetic alopecia ina female.
 4. The method of treating alopecia according to claim 1,wherein the required site of skin is the frontal region or the crown. 5.The method of treating alopecia according to claim 1, wherein there iscoexisting seborrheic alopecia or alopecia pityroides.
 6. The method oftreating alopecia according to claim 1, wherein the alopecia areata isnormal alopecia areata, alopecia totalis, alopecia universalis, oralopecia ophiasis.
 7. The method of treating alopecia according to claim1, wherein the alopecia is alopecia of a subject that-has attained asteroid-dependent state or a subject for which a steroid treatment agentcannot be used.
 8. The method of treating alopecia according to claim 1,wherein the alopecia areata is S1 or B0 alopecia or above.
 9. The methodof treating alopecia according to claim 1, wherein a therapeutic effectis obtained by application for 1 week or longer.
 10. The method oftreating alopecia according to claim 1, wherein there is no recurrencefor a period of 1 month or longer even when application is stopped. 11.The method of treating alopecia according to claim 1, wherein the dosageform of the composition is an ointment, a gel, a cream, a lotion, aliquid, a wax, a powder, a spray, a gel spray, a foam, a shampoo, atreatment, a scalp treatment, or a tonic.
 12. The method of treatingalopecia according to claim 1, wherein the content of the C-typenatriuretic peptide (CNP) in the composition is 1 to 1000 μg/g.
 13. Themethod of treating alopecia according to claim 1, wherein the C-typenatriuretic peptide (CNP) comprises CNP-22 and/or CNP-53.
 14. The methodof treating alopecia according to claim 1, wherein the alopecia istreated without causing an itching sensation.